CN102105583A - Method for collection of nucleic acid from fecal sample, method for analysis of nucleic acid, and apparatus for treatment of fecal sample - Google Patents
Method for collection of nucleic acid from fecal sample, method for analysis of nucleic acid, and apparatus for treatment of fecal sample Download PDFInfo
- Publication number
- CN102105583A CN102105583A CN2009801285392A CN200980128539A CN102105583A CN 102105583 A CN102105583 A CN 102105583A CN 2009801285392 A CN2009801285392 A CN 2009801285392A CN 200980128539 A CN200980128539 A CN 200980128539A CN 102105583 A CN102105583 A CN 102105583A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- ight soil
- soil sample
- solution
- mentioned
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 374
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 374
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 374
- 238000000034 method Methods 0.000 title claims abstract description 171
- 238000004458 analytical method Methods 0.000 title claims description 45
- 230000002550 fecal effect Effects 0.000 title abstract 7
- 230000003321 amplification Effects 0.000 claims abstract description 89
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 89
- 210000003608 fece Anatomy 0.000 claims abstract description 88
- 239000003960 organic solvent Substances 0.000 claims abstract description 86
- 239000007787 solid Substances 0.000 claims abstract description 57
- 239000002689 soil Substances 0.000 claims description 472
- 239000000523 sample Substances 0.000 claims description 247
- 230000002401 inhibitory effect Effects 0.000 claims description 101
- 239000000126 substance Substances 0.000 claims description 99
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 79
- 238000006243 chemical reaction Methods 0.000 claims description 71
- 238000011084 recovery Methods 0.000 claims description 63
- 210000004027 cell Anatomy 0.000 claims description 38
- 230000007246 mechanism Effects 0.000 claims description 35
- 239000000203 mixture Substances 0.000 claims description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 29
- 210000004962 mammalian cell Anatomy 0.000 claims description 29
- 241000305071 Enterobacterales Species 0.000 claims description 28
- 108020004414 DNA Proteins 0.000 claims description 21
- 239000000470 constituent Substances 0.000 claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 16
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 15
- 150000003839 salts Chemical class 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 14
- 239000013543 active substance Substances 0.000 claims description 13
- 210000002429 large intestine Anatomy 0.000 claims description 13
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 13
- 230000008859 change Effects 0.000 claims description 12
- 230000003196 chaotropic effect Effects 0.000 claims description 12
- 201000010099 disease Diseases 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 150000002576 ketones Chemical class 0.000 claims description 11
- 239000003550 marker Substances 0.000 claims description 9
- 238000000926 separation method Methods 0.000 claims description 8
- 239000002699 waste material Substances 0.000 claims description 7
- 230000036425 denaturation Effects 0.000 claims description 5
- 238000004925 denaturation Methods 0.000 claims description 5
- 238000009795 derivation Methods 0.000 claims description 5
- 150000002989 phenols Chemical group 0.000 claims description 5
- 108700042226 ras Genes Proteins 0.000 claims description 5
- CKMXBZGNNVIXHC-UHFFFAOYSA-L ammonium magnesium phosphate hexahydrate Chemical compound [NH4+].O.O.O.O.O.O.[Mg+2].[O-]P([O-])([O-])=O CKMXBZGNNVIXHC-UHFFFAOYSA-L 0.000 claims description 4
- 231100000504 carcinogenesis Toxicity 0.000 claims description 4
- 230000001079 digestive effect Effects 0.000 claims description 4
- 238000010195 expression analysis Methods 0.000 claims description 4
- 108020004999 messenger RNA Proteins 0.000 claims description 4
- 230000011987 methylation Effects 0.000 claims description 4
- 238000007069 methylation reaction Methods 0.000 claims description 4
- 230000035772 mutation Effects 0.000 claims description 4
- 230000010309 neoplastic transformation Effects 0.000 claims description 4
- 229910052567 struvite Inorganic materials 0.000 claims description 4
- 239000000725 suspension Substances 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 3
- 239000002683 reaction inhibitor Substances 0.000 abstract 2
- 239000004480 active ingredient Substances 0.000 abstract 1
- 238000010828 elution Methods 0.000 abstract 1
- 239000003112 inhibitor Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 194
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 73
- 239000006228 supernatant Substances 0.000 description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 28
- 210000000941 bile Anatomy 0.000 description 26
- 230000000694 effects Effects 0.000 description 26
- 230000009467 reduction Effects 0.000 description 23
- 206010028980 Neoplasm Diseases 0.000 description 22
- 238000002156 mixing Methods 0.000 description 22
- 201000011510 cancer Diseases 0.000 description 21
- 238000007689 inspection Methods 0.000 description 21
- 206010009944 Colon cancer Diseases 0.000 description 20
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 20
- 238000005119 centrifugation Methods 0.000 description 20
- 201000010989 colorectal carcinoma Diseases 0.000 description 19
- 238000004321 preservation Methods 0.000 description 15
- 230000001476 alcoholic effect Effects 0.000 description 13
- -1 pottery Substances 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 9
- 239000004743 Polypropylene Substances 0.000 description 9
- 239000003833 bile salt Substances 0.000 description 9
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 9
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 229920001155 polypropylene Polymers 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- 239000000284 extract Substances 0.000 description 8
- 238000003860 storage Methods 0.000 description 8
- 244000052616 bacterial pathogen Species 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000010839 reverse transcription Methods 0.000 description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
- 238000004159 blood analysis Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical class [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 description 5
- 235000019587 texture Nutrition 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 150000001298 alcohols Chemical class 0.000 description 4
- 230000004087 circulation Effects 0.000 description 4
- 238000002052 colonoscopy Methods 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 238000000354 decomposition reaction Methods 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000007710 freezing Methods 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- KEMQGTRYUADPNZ-UHFFFAOYSA-N heptadecanoic acid Chemical compound CCCCCCCCCCCCCCCCC(O)=O KEMQGTRYUADPNZ-UHFFFAOYSA-N 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 102000016911 Deoxyribonucleases Human genes 0.000 description 3
- 108010053770 Deoxyribonucleases Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000003398 denaturant Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000002101 lytic effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 229960001866 silicon dioxide Drugs 0.000 description 3
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000606125 Bacteroides Species 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- SGHZXLIDFTYFHQ-UHFFFAOYSA-L Brilliant Blue Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 SGHZXLIDFTYFHQ-UHFFFAOYSA-L 0.000 description 2
- 241000588923 Citrobacter Species 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 241000588914 Enterobacter Species 0.000 description 2
- 241000194033 Enterococcus Species 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- 241000186394 Eubacterium Species 0.000 description 2
- 101710113436 GTPase KRas Proteins 0.000 description 2
- 241000588748 Klebsiella Species 0.000 description 2
- 208000037062 Polyps Diseases 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 238000007844 allele-specific PCR Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000003149 assay kit Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013068 control sample Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 238000006073 displacement reaction Methods 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 208000021788 large intestine disease Diseases 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 238000007726 management method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002736 nonionic surfactant Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 244000045947 parasite Species 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000006920 protein precipitation Effects 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 229960004249 sodium acetate Drugs 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 238000005382 thermal cycling Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 101150071279 Apc gene Proteins 0.000 description 1
- 101150071146 COX2 gene Proteins 0.000 description 1
- 108091029523 CpG island Proteins 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 102100038595 Estrogen receptor Human genes 0.000 description 1
- 239000004214 Fast Green FCF Substances 0.000 description 1
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 description 1
- 208000033962 Fontaine progeroid syndrome Diseases 0.000 description 1
- 206010016952 Food poisoning Diseases 0.000 description 1
- 208000019331 Foodborne disease Diseases 0.000 description 1
- 101150112014 Gapdh gene Proteins 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241000724675 Hepatitis E virus Species 0.000 description 1
- 101000882584 Homo sapiens Estrogen receptor Proteins 0.000 description 1
- 101000864786 Homo sapiens Secreted frizzled-related protein 2 Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010022653 Intestinal haemorrhages Diseases 0.000 description 1
- 206010069755 K-ras gene mutation Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102100025825 Methylated-DNA-protein-cysteine methyltransferase Human genes 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 102000001621 Mucoproteins Human genes 0.000 description 1
- 108010093825 Mucoproteins Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- KUIFHYPNNRVEKZ-VIJRYAKMSA-N O-(N-acetyl-alpha-D-galactosaminyl)-L-threonine Chemical compound OC(=O)[C@@H](N)[C@@H](C)O[C@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1NC(C)=O KUIFHYPNNRVEKZ-VIJRYAKMSA-N 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 241000269799 Perca fluviatilis Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102100030054 Secreted frizzled-related protein 2 Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- SAQSTQBVENFSKT-UHFFFAOYSA-M TCA-sodium Chemical compound [Na+].[O-]C(=O)C(Cl)(Cl)Cl SAQSTQBVENFSKT-UHFFFAOYSA-M 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 210000000013 bile duct Anatomy 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 235000012745 brilliant blue FCF Nutrition 0.000 description 1
- 239000004161 brilliant blue FCF Substances 0.000 description 1
- 229940055580 brilliant blue fcf Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- BTANRVKWQNVYAZ-UHFFFAOYSA-N butan-2-ol Chemical class CCC(C)O BTANRVKWQNVYAZ-UHFFFAOYSA-N 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000012505 colouration Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 235000019439 ethyl acetate Nutrition 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 239000003172 expectorant agent Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 235000019240 fast green FCF Nutrition 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000003633 gene expression assay Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 230000006607 hypermethylation Effects 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- KHLVKKOJDHCJMG-QDBORUFSSA-L indigo carmine Chemical compound [Na+].[Na+].N/1C2=CC=C(S([O-])(=O)=O)C=C2C(=O)C\1=C1/NC2=CC=C(S(=O)(=O)[O-])C=C2C1=O KHLVKKOJDHCJMG-QDBORUFSSA-L 0.000 description 1
- 229960003988 indigo carmine Drugs 0.000 description 1
- 235000012738 indigotine Nutrition 0.000 description 1
- 239000004179 indigotine Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 108040008770 methylated-DNA-[protein]-cysteine S-methyltransferase activity proteins Proteins 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229940066491 mucolytics Drugs 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000001073 sample cooling Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- DCKVNWZUADLDEH-UHFFFAOYSA-N sec-butyl acetate Chemical compound CCC(C)OC(C)=O DCKVNWZUADLDEH-UHFFFAOYSA-N 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000009518 sodium iodide Nutrition 0.000 description 1
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 description 1
- 229910001488 sodium perchlorate Inorganic materials 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 235000012976 tarts Nutrition 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/101—Temperature
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2527/00—Reactions demanding special reaction conditions
- C12Q2527/125—Specific component of sample, medium or buffer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Sampling And Sample Adjustment (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Disclosed is a method for collecting a nucleic acid from a fecal sample with high purity without the need of any complicated manipulation. Also disclosed is an apparatus for treating a fecal sample for use in the method. Further disclosed is a method for analyzing by using a nucleic acid collected by the aforementioned method. The nucleic acid collection method comprises the following steps (A) to (C): (A) adding the collected feces to a nucleic acid amplification reaction inhibitor removal solution comprising a water-soluble organic solvent as an active ingredient to prepare a fecal sample, and storing the fecal sample for a predetermined period to cause the elution of a nucleic acid amplification inhibitor; (B), subsequent to the step (A), removing the nucleic acid amplification reaction inhibitor removal solution from the fecal sample to collect a solid content derived from the feces; and (C) collecting a nucleic acid from the feces-derived solid content collected in the step (B).
Description
Technical field
The present invention relates to a kind of method that from the ight soil sample, reclaims nucleic acid that is used for reclaiming nucleic acid, used the method for nucleic acid analysis and the ight soil apparatus for samples treatment of the nucleic acid that reclaims by this nucleic acid recovery method from ight soil sample high purity ground.
The application is based on requiring right of priority Japanese Patent Application 2008-189684 number of Japanese publication on July 23rd, 2008, and its content is incorporated herein.
Background technology
Same with the America and Europe, in Japan, patient's number of colorectal carcinoma is also sharply increasing year by year, and colorectal carcinoma has been occupied former positions of cancer mortality.What its reason thought that Japanese dietetic life is transformed into American-European type is the center with the meat.Particularly, suffer from colorectal carcinoma about annual about 60,000 people, aspect the death toll of internal organs classification, colorectal carcinoma comes the 3rd, is only second to cancer of the stomach, lung cancer, and prediction also can further increase from now on.On the other hand, colorectal carcinoma is different with other cancer, and by treating at the initial stage of a disease, nearly 100% can cure.Therefore, the object that colorectal carcinoma is examined as early cancer is very significant, and the research and development that is used for the inspection method of early discovery colorectal carcinoma prevails.
As the inspection method that is used for the early discovery colorectal carcinoma, that carries out has for example bowel lavage inspection, a colonoscopy etc.The bowel lavage inspection is following inspection: inject barium and make its mucosal surface that is attached to large intestine in large intestine, the irradiation X ray is taken the concavo-convex of its surface, observes the surface of large intestine.On the other hand, colonoscopy is the inspection by endoscope direct viewing large intestine inside.Particularly the sensitivity of colonoscopy and specificity height also have the advantage that also can excise polyp and early cancer.Yet the problem that the burden that there are the cost height in these inspection methods, bring to the experimenter is big, follow the risk of complication.For example, exist X ray to expose and ileac danger in the bowel lavage inspection.In addition, colonoscopy is owing to directly inserting endoscope in the large intestine thereby having invasive.And then, need endoscopic procedure skilled, the facility that can check is limited.Therefore, these inspection methods are unsuitable for having regular physical checkups etc. with the colorectal carcinoma inspection of asymptomatic common artificial object.
In recent years, as a screening method of colorectal carcinoma, just extensively implement a kind of Noninvasive and ight soil occult blood examination cheaply.Ight soil occult blood examination is the inspection that has or not of investigating the oxyphorase in red corpuscle source contained in the ight soil, and is the method for the existence of indirect predictions colorectal carcinoma.The reason that occult blood examination is widely used as ight soil can list: can carry out the collection and the preservation of ight soil at normal temperatures, also not need to refrigerate and special preservation condition such as freezing; And, can carry out simply at average family; Operate very easy.But, in ight soil occult blood examination, have the problem that sensitivity is low to moderate about 25%, the probability of omission colorectal carcinoma is high.And then positive hit rate is also low, and is actual in colorectal carcinoma patient's ratio is below 10% among the positive experimenter of ight soil occult blood examination, comprises a lot of false positives.Therefore, the higher novel inspection method of strong expectation exploitation reliability.
As also be suitable for having regular physical checkups etc., Noninvasive, easy and novel inspection method that reliability is high, in the investigation ight soil cancer-free cell is arranged or has the inspection of the gene in cancer-free cell source to be attracted attention.These inspection methods are because direct surveys have the gene in cancer-free cell or cancer cells source, therefore can expect to become with investigation have or not the ill generation of following colorectal carcinoma digestive tract hemorrhage, compare the higher test procedure of reliability as the ight soil occult blood examination method of indirect colorectal carcinoma inspection method.
In order to detect cancer cells in the ight soil etc. with high precision, the nucleic acid that reclaims the cancer cells source in the ight soil effectively is very important.Particularly, because the nucleic acid in the source of the cancer cells in the ight soil is trace, and contains a large amount of digestion residues and bacterium in the ight soil, so nucleic acid is very easy to be decomposed.Therefore, in order to reclaim the nucleic acid in mammalian cells sources such as nucleic acid, particularly people effectively from the ight soil sample, importantly modulate the ight soil sample with the decomposition that prevents the ight soil amplifying nucleic acid and till can stably be saved to checked operation the time.As the modulator approach of this ight soil sample, the method for separating the cancer cells that comes off from digestive tubes such as large intestines from the ight soil of gathering is for example arranged.By from ight soil, separating cancer cells, can suppress the proteolytic enzyme in sources such as bacterium, the influence that lytic enzymes such as DNase, RNase cause.Method as separate cancer cells from ight soil for example discloses following method: (1) is the method for isolated cell from ight soil, and it comprises the operation that (a) is cooled to ight soil the temperature that is lower than its gel zero pour; (b) ight soil is remained below the temperature of its gel zero pour, from ight soil, gather the operation of cell simultaneously (for example, with reference to patent documentation 1 so that ight soil is essentially the state that remains intact.)。In addition, disclose following method: (2) usually are being distributed to ight soil in the transportation substratum that contains protease inhibitor matter, mucolytic agent and sterilant under the surrounding temperature, separate the cell that comes off from large intestine then (for example, with reference to patent documentation 2.)。
In addition, contain in the ight soil PCR nucleic acid amplification reactions such as (Polymerase Chain Reaction) is had inhibiting material (for example bile acide and its salt etc.) (for example, with reference to non-patent literature 1.)。For example, it is reported that adult's average defecate amount is about 200～400g/ day, bile acide excretion in healthy its ight soil of people is 200～650mg/ day, in addition, under situation with the patient of ileum obstacle, the excretion of its bile acide even be 10 times of healthy people.That is, when being scaled every 1g ight soil, contain the bile acide of the 0.5mg～3.25mg that has an appointment in every 1g ight soil of healthy people, contain unsoundness people's 10 times bile acide in every 1g ight soil of patient.
On the other hand, report that also the inhibition effect of the PCR that bile salt caused produces under the concentration about 50 μ g/mL.Therefore, extract nucleic acid and when with PCR etc. it being increased, improve in order to make amplification efficiency from ight soil, expectation prevents residual (the carry over) of the inhibitory substance in the nucleic acid amplification reaction such as bile salt.
The prior art document
Patent documentation
Patent documentation 1: the flat 11-511982 communique of Japanese Unexamined Patent Application Publication
Patent documentation 2: Japanese Unexamined Patent Application Publication 2004-519202 communique
Non-patent literature
Non-patent literature 1:Wilson IG, APPLIED AND ENVIRONMENTAL MICROBIOLOGY,, the 63rd volume, the 3741st～51 page in 1997.
Summary of the invention
The problem that invention will solve
In the method for above-mentioned (1), while cool off ight soil sample isolated cell.If under the refrigerative situation, do not carry out this lock out operation, then can't obtain detected result accurately owing to the rotten grade of ight soil sample.Therefore, in order to prevent the rotten of ight soil sample effectively, after gathering ight soil, cool off very important rapidly.Yet, as examination etc. when carrying out feces collection at home,, unrealistic after the collection immediately with the unusual difficulty of ight soil sample cooling.In addition, from the ight soil sample operation of isolated cell very numerous and diverse, exist to cause the problem of checking that required expense increases.
In the method for above-mentioned (2), by adding sterilant etc., thereby do not need cooling operation just can at room temperature carry out the modulation and the preservation of ight soil sample, but owing to need from ight soil, separate the operation of the cell that comes off from large intestine, therefore operability still is poor, as a result, the problem that has the cost cost.
And then, patent documentation 1 and 2 and non-patent literature 1 in, prevent inhibitory substance residual in the nucleic acid amplification reactions such as bile acide, bile salt when all not having record from ight soil, to reclaim nucleic acid fully.
The object of the present invention is to provide a kind of reduce inhibitory substance in the nucleic acid amplification reaction such as bile acide residual, do not need numerous and diverse operation and high purity ground to reclaim the method for the nucleic acid in the ight soil; The method that the nucleic acid that use is reclaimed by this method is analyzed; And the ight soil apparatus for samples treatment that uses in this recovery and the analytical procedure.
The scheme that is used to deal with problems
The inventor etc. are in order to solve above-mentioned problem, find, by before the nucleic acid extraction operation, it is that inhibitory substance in the nucleic acid amplification reaction of effective constituent is removed in the solution of usefulness and preserved specific time that the ight soil of gathering is immersed in the water-miscible organic solvent, can stripping and remove inhibitory substance in the nucleic acid amplification reaction contained in the ight soil, high purity ground reclaims nucleic acid from ight soil.
That is, the present invention includes,
(1) a kind of method that from the ight soil sample, reclaims nucleic acid, it is the method that high purity ground reclaims nucleic acid from ight soil, has following operation:
(A) ight soil of gathering being added to the water-miscible organic solvent is that removing with in the solution of inhibitory substance in the nucleic acid amplification reaction of effective constituent modulated the ight soil sample, and above-mentioned ight soil sample preserved specific time, thereby make the operation of nucleic acid amplification reaction inhibitory substance stripping;
(B) afterwards, from the ight soil sample, remove removing with solution and reclaiming the operation of the solids component in ight soil source of inhibitory substance in the nucleic acid amplification reaction in operation (A); With
(C) operation of recovery nucleic acid from the solids component that the ight soil that reclaims operation (B) is originated.
(2) in above-mentioned (1) described method that from the ight soil sample, reclaims nucleic acid, can the ight soil of gathering not made suspension yet, remove with in the solution modulation ight soil sample but add the nucleic acid amplification reaction inhibitory substance immediately to.
(3) in the described method that reclaims nucleic acid from the ight soil sample in above-mentioned (1) or (2), the above-mentioned water-miscible organic solvent of use is preferably water-soluble alcohol and/or ketone.
(4) in the described method that reclaims nucleic acid from the ight soil sample in above-mentioned (1)～(3), the concentration of the above-mentioned water-miscible organic solvent of use is preferably more than 30%.
(5) in the described method that from the ight soil sample, reclaims nucleic acid in above-mentioned (3) or (4), the above-mentioned water-miscible organic solvent of use preferably contain be selected from the group of forming by ethanol, propyl alcohol and methyl alcohol more than a kind as water-soluble alcohol.
(6) in above-mentioned (1) described method that reclaims nucleic acid from the ight soil sample, the above-mentioned water-miscible organic solvent of use is preferably ethanol.
(7) in the described method that reclaims nucleic acid from the ight soil sample in above-mentioned (3) or (4), the above-mentioned water-miscible organic solvent of use preferably contains acetone and/or methylethylketone as ketone.
(8) in above-mentioned (1)～(7) in each described method that from the ight soil sample, reclaims nucleic acid, the mixture ratio that above-mentioned ight soil that uses and above-mentioned nucleic acid amplification reaction inhibitory substance are removed with solution is preferably: establishing faecal volume is 1 o'clock, with respect to this 1, it is more than 1 that the nucleic acid amplification reaction inhibitory substance is removed with liquor capacity.
(9) in above-mentioned (1)～(8) in each described method that reclaims nucleic acid from the ight soil sample, the nucleic acid amplification reaction inhibitory substance of removing from the ight soil sample in the above-mentioned operation (B) is removed with solution and is preferably undertaken by centrifugal separation.
(10) in above-mentioned (1)～(9) in each described method that reclaims nucleic acid from the ight soil sample, in the above-mentioned operation (A), the time of preserving the ight soil sample was preferably more than 1 hour.
(11) in above-mentioned (1)～(9) in each described method that reclaims nucleic acid from the ight soil sample, in the above-mentioned operation (A), the time of preserving the ight soil sample was preferably more than 12 hours.
(12) in above-mentioned (1)～(9) in each described method that reclaims nucleic acid from the ight soil sample, in the above-mentioned operation (A), the time of preserving the ight soil sample was preferably more than 24 hours.
(13) in above-mentioned (1)～(9) in each described method that reclaims nucleic acid from the ight soil sample, in the above-mentioned operation (A), the time of preserving the ight soil sample was preferably more than 72 hours.
(14) in above-mentioned (1)～(13) in each described method that reclaims nucleic acid from the ight soil sample, in the above-mentioned operation (A), the temperature of preserving the ight soil sample is preferably more than 4 ℃.
(15) in above-mentioned (1)～(13) in each described method that reclaims nucleic acid from the ight soil sample, in the above-mentioned operation (A), the temperature of preserving the ight soil sample is preferably more than 20 ℃.
(16) in above-mentioned (1)～(15) in each described method that reclaims nucleic acid from the ight soil sample, the above-mentioned nucleic acid amplification reaction inhibitory substance of use is removed with solution and is preferably contained tensio-active agent.
(17) in above-mentioned (1)～(16) in each described method that reclaims nucleic acid from the ight soil sample, the above-mentioned nucleic acid amplification reaction inhibitory substance of use is removed with solution and is preferably contained tinting material.
(18) each described above-mentioned operation (C) that reclaims the method for nucleic acid from the ight soil sample is preferably following operation in above-mentioned (1)～(17): recovery simultaneously often occupies the nucleic acid in enterobacteria source and the nucleic acid of the biogenetic derivation beyond the Chang Ju enterobacteria from the solids component that the ight soil that reclaims operation (B) is originated.
(19) in above-mentioned (18) described method that reclaims nucleic acid from the ight soil sample, the biology that use above-mentioned often occupies beyond the enterobacteria is preferably mammalian cell.
(20) each described above-mentioned operation (C) that reclaims the method for nucleic acid from the ight soil sample preferably has in above-mentioned (1)～(19):
(a) make protein denaturation in the solids component in the ight soil source of reclaiming in the operation (B), and make the operation that often occupy in biology enterobacteria and Chang Ju enterobacteria beyond stripping of nucleic acid from the solids component in above-mentioned ight soil source; With
(b) reclaim the operation of the nucleic acid of stripping in the above-mentioned operation (a).
(21) in above-mentioned (20) described method that from the ight soil sample, reclaims nucleic acid, preferably above-mentioned operation (a) afterwards, above-mentioned operation (b) has (c) before and removes the proteinic operation of sex change by above-mentioned operation (a).
(22) in the described method that from the ight soil sample, reclaims nucleic acid in above-mentioned (20) or (21), the proteinic sex change in the above-mentioned operation (a) preferably use a kind of being selected from the group of forming by chaotropic salt, organic solvent and tensio-active agent with on carry out.
(23) in above-mentioned (22) described method that reclaims nucleic acid from the ight soil sample, the above-mentioned organic solvent of use is preferably phenols.
(24) in above-mentioned (21)～(23) in each described method that reclaims nucleic acid from the ight soil sample, the sex change in the above-mentioned operation (c) proteinic removed preferred use chloroform and carries out.
(25) in above-mentioned (20)～(24) in each described method that reclaims nucleic acid from the ight soil sample, the recovery of the nucleic acid in the above-mentioned operation (b) preferably has following operation:
(b1) make the nucleic acid of stripping in the above-mentioned operation (a) be adsorbed in the operation of inorganic supporting body; With
(b2) make the operation of the nucleic acid of absorption in the above-mentioned operation (b1) from the inorganic supporting body stripping.
(26) in addition, the nucleic acid of the present invention for reclaiming by each described method that from the ight soil sample, reclaims nucleic acid in above-mentioned (1)～(25).
(27) in addition, the method for nucleic acid analysis that the present invention analyzes the nucleic acid in mammalian cell source for the nucleic acid that uses recovery, the nucleic acid of this recovery is for utilizing the nucleic acid that each described nucleic acid recovery method reclaims in above-mentioned (1)～(26) from the ight soil sample.
(28) in above-mentioned (27) described method for nucleic acid analysis, the above-mentioned mammalian cell of use is preferably the digestive tube cell.
(29) in above-mentioned (27) described method for nucleic acid analysis, the above-mentioned mammalian cell of use is preferably the cell that comes off from large intestine.
(30) in above-mentioned (27)～(29) in each described method for nucleic acid analysis, the nucleic acid in analyzed above-mentioned mammalian cell source is preferably the marker of indication neoplastic transformation.
(31) in above-mentioned (27)～(29) in each described method for nucleic acid analysis, the nucleic acid in analyzed above-mentioned mammalian cell source is preferably the struvite chylopoietic disease's of indication marker.
(32) in above-mentioned (27)～(31) analysis in each described method for nucleic acid analysis be preferably be selected from the group of forming by the methylation analysis of the mutation analysis of the expression analysis of mRNA, K-ras gene and DNA more than a kind.
(33) in addition, the present invention comprises that solution removes the ight soil apparatus for samples treatment of mechanism, this solution is removed mechanism and is used for removing following nucleic acid amplification reaction inhibitory substance from the ight soil sample and removes and use solution, and this ight soil sample is that the ight soil that will gather is soaked in the water-miscible organic solvent is that the nucleic acid amplification reaction inhibitory substance of effective constituent is removed with the ight soil sample of preserving in the solution behind the specific time.
(34) the above-mentioned solution in above-mentioned (33) described ight soil apparatus for samples treatment is removed mechanism and is preferably had centrifuge mechanism, solution suction output mechanism and waste liquid recoverer.
(35) in above-mentioned (34) described ight soil apparatus for samples treatment, preferred above-mentioned solution suction output mechanism is a solution suction discharge nozzle, and this ight soil apparatus for samples treatment also has the mechanism of the above-mentioned solution suction of washing discharge nozzle.
(36) the present invention removes for a kind of nucleic acid amplification reaction inhibitory substance and uses solution, and it is to be used to modulate nucleic acid to reclaim the solution of using the ight soil sample, is effective constituent with the water-miscible organic solvent, in order to reduce the nucleic acid amplification reaction inhibitory substance in the nucleic acid that reclaims.
(37) above-mentioned (36) described nucleic acid amplification reaction inhibitory substance is removed with in the solution, and above-mentioned water-miscible organic solvent is preferably water-soluble alcohol and/or ketone.
(38) above-mentioned (37) described nucleic acid amplification reaction inhibitory substance is removed with in the solution, and the concentration of above-mentioned water-miscible organic solvent is preferably more than 30%.
(39) the present invention is a kind of feces collection test kit, and it has each described nucleic acid amplification reaction inhibitory substance in above-mentioned (36)～(38) and removes with solution and contain this nucleic acid amplification reaction inhibitory substance and remove feces collection container with solution.
The effect of invention
According to the method that reclaims nucleic acid from the ight soil sample of the present invention, nucleic acid amplification reaction inhibitory substance contained in the ight soil is removed in stripping effectively, and reclaims highly purified nucleic acid from the ight soil sample.In addition, according to the present invention, owing to do not need from the ight soil sample to separate mammalian cell etc.,, also can reduce labour and cost effectively even therefore when handling a plurality of determinand as biological or its cell of detected object etc. and so on numerous and diverse operation.Particularly, the ight soil apparatus for samples treatment of the application of the invention can reclaim nucleic acid more easily from the ight soil sample.
Like this, by utilizing the method for nucleic acid and the method for nucleic acid analysis that uses the nucleic acid that reclaims by this recovery method of from the ight soil sample, reclaiming of the present invention, thus can highly sensitive and analyze nucleic acid in the ight soil accurately.Therefore, expectation is by utilizing the present invention, and is useful to early discovery and diagnosis, the observation of therapeutic process and the pathological research of other abnormal conditions etc. with various symptoms headed by the colorectal carcinoma and disease.
Description of drawings
Fig. 1 is the figure of an embodiment of expression ight soil apparatus for samples treatment of the present invention.
Fig. 2 is the figure of expression feces collection of the present invention with the embodiment of the feces collection container A that can use in the test kit.
Fig. 3 is the figure of expression feces collection of the present invention with an example of the embodiment of the feces collection container B that can use in the test kit and its using method.
Fig. 4 is the figure of the bile acide amount in the RNA solution that per 100 μ L are extracted among the expression embodiment 1.
Fig. 5 is for being transverse axis, being the figure of the measurement result of the longitudinal axis with GAPDH amplification amount with the Septochol na concn among the expression embodiment 2.
Description of reference numerals
1 ... container body
1a ... protrusion
2 ... lid
3 ... the feces collection rod
3a ... cup
S ... the nucleic acid amplification reaction inhibitory substance is removed and is used solution
11 ... container body
12 ... lid
13 ... the feces collection rod
13a ... the hole
13b ... movable cover
15 ... bag
E ... ight soil
101 ... the ight soil apparatus for samples treatment
102 ... centrifuge mechanism
103 ... solution suction discharge nozzle
104 ... the waste liquid recoverer
105 ... solution suction discharge nozzle washing mechanism
Embodiment
Among the present invention, the nucleic acid amplification reaction inhibitory substance is meant has inhibiting material to nucleic acid amplification reaction, particularly, can list bile acide, bile salt etc.Below, abbreviate the nucleic acid amplification reaction inhibitory substance as inhibitory substance A.In addition, among the present invention, nucleic acid amplification reaction means utilizing archaeal dna polymerase and following the amplified reaction of nucleic acid elongation as PCR etc.
The method that from the ight soil sample, reclaims nucleic acid of the present invention (below, be called " nucleic acid recovery method of the present invention " sometimes.), with the ight soil gathered with the water-miscible organic solvent be effective constituent inhibitory substance A remove mix with solution after, the preservation specific time.By with ight soil with inhibitory substance A remove with solution blended state under preserve specific time, inhibitory substance A such as bile acide contained in the ight soil and bile salt are removed in stripping effectively.Thus, inhibitory substance A residual in the nucleic acid that from the ight soil sample, reclaims can be significantly reduced, highly purified nucleic acid can be reclaimed.
Inferring the reduction effect of the above-mentioned inhibitory substance A amount that water-miscible organic solvent brought, promptly can make the effect of the residual minimizing of inhibitory substance A in the nucleic acid that reclaims, is that the deliquescent difference in water-miscible organic solvent causes by inhibitory substance A and nucleic acid.That is, inhibitory substance A such as bile acide are soluble in the water-miscible organic solvents such as alcohol, but are insoluble in the non-polar organic solvent.On the other hand, nucleic acid because the effect of salt and insoluble, is insoluble in the water-miscible organic solvent such as alcohol in alcohol.Therefore infer, if ight soil is mixed with water-miscible organic solvents such as alcohol, then can make inhibitory substance A stripping in water-miscible organic solvent,, thereby can reduce inhibitory substance A amount in the nucleic acid of recovery the separate nucleic acid of itself and water insoluble solubleness organic solvent composition.
About nucleic acid recovery method of the present invention, particularly, at first, as operation (A), it is that the nucleic acid amplification reaction inhibitory substance of effective constituent is removed with in the solution (below, abbreviate as to remove and use solution S) that the ight soil of gathering is added to the water-miscible organic solvent, modulates the ight soil sample.Then, synthetic above-mentioned ight soil sample is preserved specific time, thereby make inhibitory substance A stripping.
Removing with solution S of using in the nucleic acid recovery method of the present invention is effective constituent with water-miscible organic solvent.Biological materials such as ight soil contain a large amount of moisture usually, but remove with solution S with the high solvent of the solubleness in water or can be with water with arbitrary proportion blended water-miscible organic solvent as effective constituent.Therefore, can mix rapidly, obtain the reduction effect of higher inhibitory substance A amount with the ight soil sample.
Among the present invention, water-miscible organic solvent is meant alcohols or ketone, is to have linear chain structure and for example be liquid solvents 15～40 ℃ times near room temperature.In addition, do not contain organic acid in the water-miscible organic solvent among the present invention.By with water-miscible organic solvent with linear chain structure as effective constituent, and compare as effective constituent with organic solvent with ring texturees such as phenyl ring, can promptly carry out and the mixing of ight soil.Have the easy usually and water sepn of organic solvent of ring texture, therefore be difficult for mixing the reduction effect of the inhibitory substance A amount that is difficult to obtain with ight soil.Even the organic solvent of dissolved with ring texture to a certain degree in water disperses equably in order to make ight soil, also need acutely to mix or heat.Also consider in addition,, make the mixing solutions of organic solvent and water in advance, then ight soil is mixed with this mixing solutions in order to be easy to that ight soil is mixed with the organic solvent with ring texture.Yet in order to make this mixing solutions, the organic solvent that needs to have ring texture acutely mixes with water or heats, so not preferred.
Removing and being preferably solubleness in water with water-miscible organic solvent contained in the solution S is water-miscible organic solvent more than the 12 weight %, more preferably the solubleness in water is the water-miscible organic solvent more than the 20 weight %, more preferably the solubleness in water is the water-miscible organic solvent more than the 90 weight %, be preferably especially can with water with arbitrary proportion blended water-miscible organic solvent.As can with water with arbitrary proportion blended water-miscible organic solvent, methyl alcohol, ethanol, n-propyl alcohol, 2-propyl alcohol, acetone etc. are for example arranged.
Remove with water-miscible organic solvent contained in the solution S so long as satisfy the low but inhibitory substance A of the solvability of above-mentioned definition, nucleic acid can the dissolved solvent, then be not particularly limited.As this water-miscible organic solvent, alcohols and ketone are for example arranged, as alcohols methyl alcohol as water-soluble alcohol, ethanol, propyl alcohol, butanols, mercaptoethanol etc. are arranged, as ketone acetone, methylethylketone (solubleness in water is 90 weight %) etc. are arranged.Propyl alcohol can be n-propyl alcohol, also can be the 2-propyl alcohol.In addition, butanols can be 1-butanols (solubleness in water is 20 weight %), also can be 2-butanols (solubleness in water is 12.5 weight %).
As removing with contained water-miscible organic solvent in the solution S, preferred alcohols or ketone, more preferably be selected from the group of forming by water-soluble alcohol, acetone and methylethylketone more than a kind.In these solvents, the solvability height of inhibitory substance A such as bile salt can obtain the reduction effect that more excellent inhibitory substance A measures.And then, from easily acquired, handle viewpoints such as easiness, security, more preferably water-soluble alcohol, further preferred alcohol, propyl alcohol, methyl alcohol.Ethanol particularly because security is the highest, is also handled within the family easily, and is therefore particularly useful in screening test such as have regular physical checkups.
Remove with the water-miscible organic solvent concentration in the solution S as long as be the concentration of the reduction effect that can bring into play inhibitory substance A amount, then be not particularly limited, can consider the kind etc. of water-miscible organic solvent and suitably decision.For example, when using water-soluble alcohol, ketone as effective constituent, the water-miscible organic solvent concentration of removing with solution S is preferably more than 30%.By making water-miscible organic solvent concentration is sufficient high density, thereby mixes ight soil and remove when using solution S, and the water-miscible organic solvent composition promptly is penetrated in the ight soil, can bring into play the effect of removing of inhibitory substance A rapidly.
In addition, in the present invention and the present specification, short of special record, " % " means " volume % ".
When particularly using water-soluble alcohol as effective constituent, the water-miscible organic solvent concentration of removing with solution S is preferably more than 30%, more preferably more than 50%, and more preferably 50～80%, be preferably more than 50% especially and less than 70%.If even water-miscible organic solvent concentration height then for the many ight soil of moisture content, is removed with solution S also enough with a spot of, and can bring into play the reduction effect that sufficient inhibitory substance A measures.
In addition, when using acetone, methylethylketone as effective constituent, water-miscible organic solvent concentration of removing with solution S of the present invention is preferably more than 30%, more preferably more than 60%, more preferably more than 80%.
In addition, the water-miscible organic solvent that uses among the present invention can only contain a kind of water-miscible organic solvent, also can be the mixing solutions of the water-miscible organic solvent more than 2 kinds.For example, can be the mixing solutions of the alcohol more than 2 kinds, also can be the mixing solutions of the water-miscible organic solvent of pure and mild other kinds.And then, from improving lowering efficiency and nucleic acid organic efficiency aspect of inhibitory substance A amount more, also be preferably the mixing solutions of pure and mild acetone.
The volume of removing with solution S that is added in the ight soil of gathering is not particularly limited, but ight soil and the mixture ratio removed with solution S be preferably, and establishing faecal volume is 1 o'clock, with respect to this 1, the volume of removing with solution S is more than 1.This be because, remove when adding ight soil in the feces collection container of solution S to being equipped with, use solution S if use with above the removing of ight soil equivalent, ight soil is soaked in fully remove and uses in the solution S, can obtain effect of the present invention effectively.For example, ight soil can make the feces collection container lightweight and the miniaturization of removing with solution S is housed when removing with solution S equivalent.On the other hand, be 5 times with respect to ight soil and use solution S with removing of upper volume by adding, can be rapidly and carry out the dispersion of ight soil in removing the usefulness solution S effectively.And then, use solution S by using removing of this volume, the reduction of the water-miscible organic solvent concentration that moisture caused that can also suppress to contain in the ight soil.Obtain balance preferably in order to make to be equipped with to remove with the lightweight of the feces collection container of solution S and this two aspects effect of raising of ight soil dispersiveness, more preferably ight soil is 1: 1～1: 20 with the mixture ratio of removing with solution S, more preferably 1: 3～1: 10, more preferably about 1: 5.
In addition, need only to the ight soil of animal, then be not particularly limited, but be preferably the ight soil that Mammals is originated, more preferably the ight soil of originating for the people for ight soil in nucleic acid recovery method of the present invention.For example, preferably in order to have regular physical checkups or diagnosis etc. and the people's that gathers ight soil, but also can be the ight soil of domestic animal or wildlife etc.In addition, can be the ight soil of having preserved certain hour after gathering as the ight soil of sample, but preferred ight soil after just having gathered.And then the ight soil of collection is preferably the ight soil after the firm drainage, but also can be to drain after ight soil after a while.
Confession is not particularly limited in the amount of the ight soil of nucleic acid recovery method of the present invention, but is preferably the scope of 10mg～1g.Therefore when the ight soil amount was too much, acquisition operations was taken time and energy, and feces collection container also becomes greatly, and the property handled etc. may reduce.When opposite ight soil amount was too a small amount of, the mammalian cell numbers such as cell that come off from large intestine contained in the ight soil became very few, therefore can't reclaim required nucleic acid amount, and the precision of target nucleic acid analysis may reduce.In addition, because ight soil is inhomogeneous, promptly various uneven components ground exists, therefore for fear of the partial influence of being present in of mammalian cell, preferably when gathering ight soil from a plurality of different sites collections of ight soil.
Thereby remove and by suitable dilution water solubleness organic solvent and it to be adjusted to desired concentration with solution S and to obtain.The solvent that is used to dilute is not particularly limited, and is preferably damping fluids such as water or PBS.In addition, only otherwise the infringement water-miscible organic solvent becomes the reduction effect of the inhibitory substance A amount that branch brings, remove with except that water-miscible organic solvent, containing composition arbitrarily in the solution S.For example, chaotropic salt can be contained, also tensio-active agent can be contained.By containing chaotropic salt or tensio-active agent, thereby can more effectively suppress the cytoactive in the ight soil and the enzymic activity of various lytic enzymes.
As removing, Guanidinium hydrochloride, guanidinium isothiocyanate, sodium iodide, sodium perchlorate and sodium trichloroacetate etc. are for example arranged with contained chaotropic salt in the solution S.As removing, be preferably nonionic surfactant with contained tensio-active agent in the solution S.As this nonionic surfactant, Tween80, CHAPS (3-[3-courage amido propyl dimethylamino]-1-propanesulfonic acid inner salt), Triton X-100, Tween20 etc. are for example arranged.The kind of chaotropic salt and tensio-active agent and concentration so long as can obtain the concentration of the reduction effect of inhibitory substance A amount, then are not particularly limited, and can consider ight soil amount, operation afterwards and analytical procedure etc. and suitably decision.
In addition, remove with adding the adequate colouration agent in the solution S.By painted, can obtain to prevent that mistake from gulping down, alleviating effects such as stool colour to removing with solution S.As this tinting material, be preferably used as the colouring matter of foodstuff additive, preferred blueness or green etc.Can list as fast green FCF (green No. 3), brilliant blue FCF (blue No. 1), indigo carmine (blue No. 2) etc.In addition, can mix and add multiple tinting material, also can add separately.
Removing with after adding ight soil in the solution S, mixing with removing, can obtain the reduction effect that better inhibitory substance A measures with solution S by making ight soil.Preferred ight soil with remove with can fully carrying out mixing of solution S and the feces collection person can easy method of carrying out.This be because, remove with in the solution S by ight soil is scattered in fully, the water-miscible organic solvent composition is penetrated in the ight soil fully, and can make in the ight soil inhibitory substance A fully stripping to removing with in the solution S.In addition, mix ight soil and remove with the method for solution S so long as carry out the blended method, then be not particularly limited by physical method.For example, can be by removing with dropping into the ight soil of gathering in the sealable container of solution S to being equipped with in advance, airtight after, this container turned upside down and mix, also can mix by this container being placed on the oscillator such as vortex mixer.In addition, ight soil can also be mixed mixing with in the presence of the particulate with removing with solution S.In order to mix rapidly and fully, this blending means is preferably used the method for oscillator and is used and mix the particulate method of using.Particularly, use the particulate feces collection container, thereby in family etc. does not have the environment of special device, also can mix easy and fully by using to contain in advance to mix.
Use particle as mixing, so long as do not damage the composition that water-miscible organic solvent becomes the reduction effect of the inhibitory substance A amount that branch brings, thereby and have by can be rapidly and ight soil is dispersed to removes with the hardness in the solution S and the particle of proportion fully with ight soil collision, then be not particularly limited.And then mixing with particle can be the particle that is formed by a kind of material, also can be the particle that is formed by the material more than 2 kinds.Mixing particle as such for example has the particle that is formed by glass, pottery, plastics, latex, metal etc.In addition, mixing with particle can be magnetic-particle, also can be non-magnetic particle.
Ight soil added to remove, be in and ight soil be soaked in remove, thereby can bring into play the reduction effect of inhibitory substance A amount with the state in the solution S with in the solution S.
Therefore, not necessarily need add to ight soil remove with solution S in after ight soil is mixed with removing with solution S.Can also be in the state that ight soil is soaked in remove with in the solution S, thereby when preserving, utilize the vibration in this transportation under the situation of transportation and mix.
Like this, in order to make inhibitory substance A stripping from ight soil,, removes in ight soil with the ight soil sample preservation specific time that obtains in the solution S with being soaked in.Preserve the time of ight soil sample so long as can bring into play the time of the reduction effect of inhibitory substance A amount, then be not particularly limited, can consider kind and concentration, the ight soil of water-miscible organic solvent and remove with the ratio of mixture of solution S, storage temperature etc. suitably decision.In the nucleic acid recovery method of the present invention, the shelf time of this ight soil sample preferably preserved more than 1 hour, more preferably preserved more than 12 hours, further preferably preserved more than 24 hours, especially preferably preserved more than 72 hours.In addition, can also preserve more than 168 hours.For example, by this ight soil sample is preserved more than 1 hour at least, thereby make the amount of the inhibitory substance A of stripping reach following degree: under common PCR reaction conditions, can fully suppress the residual influence that causes of inhibitory substance A in the ight soil.
About the reduction effect of inhibitory substance A that water-miscible organic solvent brought amount, when the temperature of preserving the ight soil sample is low temperature, the effect that more can obtain when high not as temperature.Particularly, among the present invention, the storage temperature of the ight soil sample in the operation (A) is preferably more than 4 ℃, more preferably more than 20 ℃.But, preferably preserve below 50 ℃ at storage temperature.Its reason is that by preserving for a long time under the hot conditions more than 50 ℃, the concentration of the water-miscible organic solvent in this ight soil sample may wait the required sufficient concentration of reduction effect that is lower than performance inhibitory substance A amount owing to volatilization.
In the nucleic acid recovery method of the present invention, as long as the storage temperature of ight soil sample is the reduction effect that can bring into play inhibitory substance A amount more than 4 ℃.That is, the preservation in the operation (A) can use thermostat etc. to carry out under the controlled environment of temperature, but also can not need the controlled special environment of temperature, and at room temperature carries out.In addition, can also under the temperature of transportation of the collection of carrying out ight soil usually or ight soil sample etc., carry out.Therefore, for example, even when under the non-control of temperature, transporting in the operation (A) synthetic ight soil sample, also can be with should be as between preservation period between the delivery period.More specifically, under situations such as picture has regular physical checkups, the place that the feces collection person modulates the ight soil sample separates with the place of carrying out the nucleic acid extraction operation, when synthetic ight soil sample is transported to the place of carrying out the nucleic acid extraction operation, as long as the temperature during transportation is 4～50 ℃, then no matter have or not temperature control, all can be with this haulage time as the shelf time in the operation (A).
In order to reduce the amount of the inhibitory substance A from the nucleic acid that ight soil reclaims, can also after extracting nucleic acid, carry out the stripping of inhibitory substance A and remove operation.Yet when carrying out the inspection of determinand clinically, the increase of this inspection operation is directly connected to the increase of labor cost.
With respect to this, when using nucleic acid recovery method of the present invention, can carry out following operation before checking the inspection operation that carry out in the place: the feces collection person directly is soaked in ight soil after gathering ight soil and removes with in the solution S, and inhibitory substance is removed in stripping.Therefore, nucleic acid recovery method of the present invention can expect to cut down the cost in clinical examination etc.
In addition, as mentioned above, the nucleic acid in the ight soil is very easy to decompose.Therefore, modulate the ight soil sample usually after, promptly for reclaim in nucleic acid, in the analysis procedure.In addition, under the situation that ight soil sample when modulation and nucleic acid reclaim, the timed interval when analyzing is long, in order to suppress the carrying out that nucleic acid decomposes, and under low temperature environments such as freezing, refrigeration, preserve the ight soil sample.Yet, in the nucleic acid recovery method of the present invention, behind the modulation ight soil sample, even also can from this ight soil sample, reclaim nucleic acid very effectively during long-time the preservation under than higher environment at the room temperature equitemperature.This be because, by ight soil is mixed with water-miscible organic solvent, thereby prevent the decomposition etc. of nucleic acid contained in the ight soil and stably keep nucleic acid, infer that water-miscible organic solvent brought this reclaim efficiently nucleic acid, promptly prevent nucleic acid decomposition etc., stably keep and reclaim this effect of nucleic acid effectively, be because following former thereby obtain:
(1) cell that often occupies enterobacteria biology in addition that the dehydration that has owing to the water-miscible organic solvent composition, mammalian cell or virus etc. have as the nucleic acid of detected object is activated;
(2) since the cytoactive that often occupies enterobacteria significantly reduce, and through the time variation be suppressed;
(3) denaturation of protein that has owing to the water-miscible organic solvent composition, the active of various lytic enzymes such as the proteolytic enzyme in the ight soil, DNase, RNase significantly reduces.
That is, in the nucleic acid recovery method of the present invention,, use with water-miscible organic solvent and use solution S as removing of effective constituent in order to modulate the ight soil sample.Therefore, even synthetic ight soil sample is being preserved under the situation of adequate time, also can between the preservation period of ight soil sample, suppress the decomposition of ight soil sample amplifying nucleic acid and stably keep this nucleic acid for stripping inhibitory substance A.Therefore, can after operation (C) in reclaim nucleic acid very effectively.
Inhibitory substance A contained in the ight soil preferentially is dissolved in removing with solution S, but in removing the solids component that residues in the ight soil source with the lower nucleic acid of the solvability in the solution S.Therefore, in operation (A) afterwards,, from the ight soil sample, remove and remove with solution S and reclaim the solids component in ight soil source as operation (B).Then, as operation (C), from the solids component that the ight soil that reclaims is originated, reclaim nucleic acid, thereby can reclaim the lower highly purified nucleic acid of content of inhibitory substance A.
To remove the method for from the ight soil sample, removing in the operation (B), and suitably select the common employed separation method in the time of can be from separating liquid composition and solids component and carry out with solution S.This separation method is not so long as damage nucleic acid in the ight soil sample, and the liquid component of ight soil sample promptly can be removed with solution S is isolating method the solids component in ight soil source together with the inhibitory substance A of stripping from solids component, then is not particularly limited.For example, can remove supernatant by centrifugal separation, reclaim solids component, also can utilize filtration method that the ight soil sample is carried out the solids component that strainer filters, reclaims the ight soil source that remains on the filtering surface as sedimentary ight soil source.Preferred especially centrifugal separation among the present invention.
Nucleic acid recovery method in the operation (C) is not particularly limited, so long as the method that adopts usually when reclaiming nucleic acid from sample then can be undertaken by any means.The nucleic acid that reclaims from the solids component in ight soil source both can be DNA, can be RNA, also can be DNA and RNA the two.
Mainly containing mammalian cell etc. in the solids component in ight soil source often occupies the nucleic acid in biology beyond the enterobacteria (below, be called mammalian cell) source and often occupies the nucleic acid that enterobacteria is originated.When from the solids component in ight soil source, reclaiming nucleic acid, can reclaim the nucleic acid in mammalian cell source and the nucleic acid in Chang Ju enterobacteria cell source respectively, but especially preferably reclaim simultaneously.The nucleic acid by reclaiming mammalian cell source simultaneously and the nucleic acid in Chang Ju enterobacteria source, thus a large amount of nucleic acid that often occupy the enterobacteria source that exist are brought into play function as carrier in the ight soil.Its result compares with back recovery nucleic acid such as separating mammalian cell in advance from ight soil, and the nucleic acid of originating for the less mammalian cell of amount can more effectively reclaim nucleic acid.
For example, as operation (a), make the protein denaturation in the solids component in ight soil source, make the mammalian cell of nucleic acid from the solids component in above-mentioned ight soil source etc. and often occupy stripping in the enterobacteria, then as operation (b), reclaim the nucleic acid of stripping, thereby can from the solids component in ight soil source, reclaim the nucleic acid in mammalian cell source and the nucleic acid in Chang Ju enterobacteria source simultaneously.
Proteinic sex change in the solids component in the ight soil source in the operation (a) can be undertaken by known method.For example, by in the solids component in ight soil source, adding chaotropic salt, organic solvent, tensio-active agent etc., can make the protein denaturation in the solids component in ight soil source usually as the compound of protein denaturant.In the operation (a),, can use and remove the same material of enumerating with the chaotropic salt in the solution S and tensio-active agent of material as adding to as chaotropic salt or the tensio-active agent in the solids component that adds ight soil source to.As organic solvent, be preferably phenols.Phenols can be neutrality, also can be acidity.When using the tart phenols, can optionally RNA be had precedence over DNA extraction to water layer.In addition, in the operation (a), when in the solids component in ight soil source, adding chaotropic salt, organic solvent, tensio-active agent etc., can add a kind of compound, also can add the compound more than 2 kinds.
Also can between operation (a) and operation (b), operation (c) be set, remove the protein of sex change by operation (a).By before reclaiming nucleic acid, removing the protein of sex change in advance, can improve the quality of the nucleic acid of recovery.Proteinic removing in the operation (c) can be undertaken by known method.For example, make the denatured protein precipitation and only reclaim supernatant, can remove denatured protein by centrifugation.In addition, also can add chloroform, carry out centrifugation after fully mixing by vortex mixer etc., make the denatured protein precipitation and only reclaim supernatant.Thus, compare, can remove denatured protein more completely with only carrying out centrifugation.
The recovery of the nucleic acid of stripping can be undertaken by known method such as ethanol precipitation or cesium chloride ultracentrifugations in the operation (b).As the example of recovery method, can list following operation (b1) and operation (b2).As operation (b1), the nucleic acid of stripping in the operation (a) is adsorbed on the inorganic supporting body.Then, as operation (b2), the nucleic acid that makes absorption in the operation (b1) stripping on the inorganic supporting body.Thus, can reclaim nucleic acid.The inorganic supporting body of using in the operation (b1) can be used the known inorganic supporting body that can adsorb nucleic acid.In addition, the shape of this inorganic supporting body also is not particularly limited, and can be particulate state, also can be for membranaceous.As this inorganic supporting body, silica containing particles (pearl) such as silica gel, silicon-base oxide, glass, diatomite are for example arranged, or the porous film of nylon, polycarbonate, polyacrylic ester, Nitrocellulose etc. etc.
The solvent that uses in the operation (b2) can be considered the kind of the nucleic acid that reclaims and afterwards method for nucleic acid analysis etc. and suitably use and be generally used for making the solvent of nucleic acid from these known inorganic supporting body strippings.For example, as this stripping solvent, preferred especially purified water.In addition, preferably operation (b1) afterwards, operation (b2) before, use suitable lavation buffer solution that the inorganic supporting body that is adsorbed with nucleic acid is washed.
Use contains removing of the chaotropic salt that is useful on the full concentration of stripping nucleic acid from mammalian cell etc. or tensio-active agent when modulating the solids component in ight soil source with solution S, when the solids component of originating from ight soil in the operation (C) reclaims nucleic acid, also can omit operation (a).
Directly add protein denaturants such as chaotropic salt in the solids component in the ight soil source that can in operation (B), reclaim, but add protein denaturant again after preferably temporarily being suspended in the suitable stripping with medicament.When reclaiming DNA,, for example can use phosphoric acid buffer or tris damping fluid etc. as this stripping with medicament.Preferably make the medicament of DNase inactivation, and then more preferably contain the medicament of proteolytic enzymes such as Proteinase K by high pressure steam sterilization etc.On the other hand, when reclaiming RNA,, for example can use citrate buffer solution etc. as this stripping with medicament.Yet, because RNA is the material that decomposes very easily, the therefore preferred damping fluid that contains RNase inhibitor such as guanidine thiocyanate or Guanidinium hydrochloride that uses.In addition, also can be before in operation (C), use suitable damping fluid such as PBS (phosphate buffer normal saline, pH7.4) that the solids component in the ight soil source of reclaiming in the operation (B) is washed in advance.
Difference according to analytical procedure afterwards, can from the solids component in ight soil source, not extract and purification of nucleic acid yet, and only in the solids component in ight soil source, add and mix suitable stripping with medicament, stripping nucleic acid from the solids component in ight soil source, thus nucleic acid can be reclaimed.For example, when having a large amount of pathogenic bacterias etc. in the ight soil sample, when the nucleic acid in these pathogenic bacterias sources is analyzed, from the ight soil sample, only reclaim solid form divide after, add and also mix the stripping with medicament such as PBS that contain proteolytic ferments such as Proteinase K.The uniform ight soil sample solution that obtains thus is directly used in foranalysis of nucleic acids, thereby can detects the gene etc. in pathogenic bacteria source.In addition, reclaiming nucleic acid from the solids component in ight soil source also can use commercially available test kits such as nucleic acid extraction kit, virus detection kit to carry out.
In the nucleic acid recovery method of the present invention, it is that removing with the operation of preserving the specified time in the solution S of effective constituent is assigned in preservation or the haulage time that the ight soil that will gather is soaked in the water-miscible organic solvent.Therefore, the nucleic acid recovery method of the application of the invention reclaims nucleic acid from the ight soil of gathering, thereby as screening tests such as examinations, when a spacer segment being arranged till during to foranalysis of nucleic acids behind the feces collection, when perhaps the feces collection place separates with the foranalysis of nucleic acids place, also can stably keep nucleic acid.In addition, meanwhile, can the limit be preserved or transportation ight soil sample by inhibitory substance A stripping limit.In addition, do not need to set and be used to refrigerate or machine or storage temperature condition that refrigerated is special, and can make inhibitory substance A stripping in the ight soil sample easily.
About the operation (B) in the nucleic acid recovery method of the present invention, for example, to remove the treatment unit of mechanism easy and promptly carry out thereby can contain solution by use, and this solution is removed mechanism and is used for removing as removing of liquid component from the ight soil sample and uses solution S.This solution is removed mechanism so long as usually can the separate solid composition and the solid-liquid separation mechanism of liquid component, then is not particularly limited, and is preferably centrifuge mechanism.In addition, remove by the solution suction output mechanism of the isolating supernatant of centrifuge mechanism and the device of waste liquid recoverer, then can carry out operation (B) automatically a plurality of ight soil samples so long as possess to be used to aspirate.As solution suction output mechanism, be preferably from the solution suction discharge nozzle of the nozzle suction supernatant of front end.As this solution suction discharge nozzle, can list as electric pipette etc.
Has device as the solution suction discharge nozzle of solution suction output mechanism, when it further possesses the mechanism of washing soln suction discharge nozzle, when supernatant is removed in suction from a plurality of ight soil samples, can be at each ight soil sample washing soln suction discharge nozzle, thereby can avoid outside assorted bacterium etc. to sneak in (pollution) ight soil sample.In addition, the leading section of solution suction discharge nozzle is during for the tip that can load and unload etc., for example, install and unloading mechanism by having widely used tip, can automatically replace tip etc. at each ight soil sample, even thereby under the situation that does not possess solution suction discharge nozzle washing mechanism, also can avoid pollution to the ight soil sample.
Fig. 1 is used for carrying out automatically the figure of an embodiment of the ight soil apparatus for samples treatment of operation (B) in the nucleic acid recovery method of the present invention for expression.The ight soil apparatus for samples treatment 101 of present embodiment has centrifuge mechanism 102, is used to aspirate solution suction discharge nozzle 103, waste liquid recoverer 104 and the solution suction discharge nozzle washing mechanism 105 of removing by centrifuge mechanism 102 isolating supernatants.At first, the ight soil of gathering is soaked in removing in the feces collection container with in the solution S and preserve specific time.The state that the ight soil sample is opened with the lid of feces collection container is arranged in the centrifuge mechanism 102 of this ight soil apparatus for samples treatment 101.At this moment, a plurality of ight soil samples can disposablely be set and carry out centrifugation.Then, the supernatant of the ight soil sample after the front end that makes solution suction discharge nozzle 103 and the centrifugation contacts, and aspirates and remove supernatant from feces collection container.To discard in waste liquid recoverer 104 by the supernatant that solution aspirates discharge nozzle 103 suction, then by having contacted the position of supernatant in the solution suction discharge nozzle washing mechanism 105 washing solns suction discharge nozzle.From other ight soil sample, aspirate in the same way after the washing and remove supernatant.Like this, possess the ight soil apparatus for samples treatment of mechanism shown in Figure 1, remove supernatant, can carry out operation (B) automatically from through a plurality of ight soil samples that centrifugation was handled, aspirating successively simultaneously by use.
Utilize nucleic acid recovery method of the present invention can reclaim remarkable residual, the highly purified nucleic acid that has reduced inhibitory substance A such as bile acide effectively.Therefore, can expect by the nucleic acid analysis of using nucleic acid recovery method of the present invention to reclaim is obtained the high analytical results of reliability.Therefore, the nucleic acid that utilizes nucleic acid recovery method of the present invention to reclaim can supply in various foranalysis of nucleic acids.Particularly, not only be highly suitable for the analysis of a large amount of nucleic acid that often occupy the enterobacteria source that exist in the ight soil, the analysis of the nucleic acid that often occupies enterobacteria biogenetic derivation in addition that also is highly suitable for only containing on a small quantity in the ight soil.
In addition, in the operation of nucleic acid recovery method of the present invention (C), when reclaiming mammalian cell etc. simultaneously and often occupying the nucleic acid in the nucleic acid of the biogenetic derivation beyond the enterobacteria and Chang Ju enterobacteria source, can be very effectively and high purity ground reclaim nucleic acid more than the mammalian cell source of the nucleic acid trace that often occupies the enterobacteria source.Therefore, carry out foranalysis of nucleic acids by the nucleic acid that uses such recovery, can highly sensitive and detect specific disease markers such as colorectal carcinoma accurately.
As this nucleic acid that often occupies enterobacteria biogenetic derivation in addition, for example, the nucleic acid in the pathogenic bacterium source of the initial stage of infection diseases such as the nucleic acid that the mammalian cells such as nucleic acid that have cancer cells to originate are originated, hepatitis virus or this infection disease in later stage etc.In addition, also can be the nucleic acid in parasite source.Particularly, owing to be the nucleic acid that from ight soil, reclaims, therefore the nucleic acid that utilizes nucleic acid recovery method of the present invention to reclaim preferably supplies in the analysis of the nucleic acid in digestive tube cells such as large intestine, small intestine, stomach source, but more preferably supplies in the analysis of the nucleic acid in the cell source that comes off from large intestine.
In addition, among the present invention, often occupy enterobacteria and be in the ight soil, mean to perch usually in the intestines of animals such as people, often to occupy bacterium than the bacterial cell of relatively large existence.Often occupy enterobacteria as this, Bacteroides (Bacteroides), eubacterium (Eubacterium), genus bifidobacterium (Bifidobacterium), genus clostridium obligatory anaerobic bacterias such as (Clostridium) are for example arranged, Escherichia (Escherichia), enterobacter (Enterobacter), Klebsiella (Klebsiella), Citrobacter (Citrobacter), enterococcus spp facultative anaerobes such as (Enterococcus) etc.
In addition, the nucleic acid that reclaims by nucleic acid recovery method of the present invention also be applicable to early discovery and accuracy require stronger, be used to investigate the morbidity that has or not cancer or suffer from the foranalysis of nucleic acids that infects disease.In addition, also preferred the confession in the foranalysis of nucleic acids that is used to investigate the morbidity that has or not diseases associated with inflammation such as colitis, enteritis, gastritis, pancreatitis.In addition, can also be in the inspection of the disease of large intestines such as the inspection of protrusion lesions such as polyp or stomach ulcer, small intestine, stomach, liver, gall-bladder, bile duct.
For example, by the nucleic acid that uses the nucleic acid that reclaims by nucleic acid recovery method of the present invention to detect and analyze the nucleic acid in cancer cells source, promptly undergo mutation etc., can check the morbidity of cancers such as having or not colorectal carcinoma or carcinoma of the pancreas.In addition, whether can detect the nucleic acid as the pathogenic bacteria source of infecting reasons such as disease, the nucleic acid of for example viral source or the nucleic acid in parasite source etc. by inquiry, can investigate to have or not and infect suffering from or having or not parasitic existence of disease.Particularly, detect the nucleic acid that first type, hepatitis E virus etc. are discharged to the pathogenic bacteria source in the ight soil by using the nucleic acid that reclaims by nucleic acid recovery method of the present invention, can Noninvasive and infect disease easily and check.In addition, whether can detect the pathogenetic bacteria that often occupies beyond the enterobacteria, for example intestinal bleeding property intestinal bacteria O-157 etc. by inquiry and cause the bacterium of food poisoning or the nucleic acid in pathogenic bacteria source, can investigate and have or not suffering from of microbism.
Especially preferably detect the marker of indication neoplastic transformation and the struvite chylopoietic disease's of indication marker by foranalysis of nucleic acids.Marker as this indication neoplastic transformation for example has carcinomebryonic antigen (CEA), sialic acid Tn antigen known cancer markers such as (STN), or has or not sudden change etc. in the apc gene, p53 gene, K-ras gene etc.In addition, p16, hMLHI, MGMT, p14, APC, E-calcium are mucoprotein, the isogenic methylated detection of ESR1, SFRP2 also is useful (for example, with reference to Lind et al., " A CpG island hypermethylation profile of primary colorectal carcinomas andcolon cancer cell lines ", Molecular Cancer,, the 3rd volume the 28th chapter in 2004) as the diagnostic marker of large intestine disease.In addition, the DNA that has reported the Hp source in the ight soil sample is used as gastric cancer marker thing (for example with reference to No. 8, the 3781st～8 page of Nilsson et al., Journal of ClinicalMicrobiology,, the 42nd volume in 2004).On the other hand, the marker as the struvite chylopoietic disease of indication for example has the nucleic acid of Cox-2 gene source etc.
The nucleic acid that reclaims from the ight soil sample of the present invention can adopt known method for nucleic acid analysis to analyze.As this method for nucleic acid analysis, the method using PCR to wait to detect specific base sequence zone is for example arranged and nucleic acid is carried out quantitative methods etc.For example, have or not transgenation, can investigate the morbidity that has or not cancer by the base sequence zone of detecting coding oncogene etc. and the base sequence zone of containing microsatellite etc.During DNA that use is reclaimed from the ight soil sample, can test example such as DNA on methylate, the sudden changes such as insertion, disappearance, displacement, repetition or inversion of base.In addition, when reclaiming RNA, behind the synthetic cDNA of reverse transcription reaction (RT:Reverse transcription), this cDNA and DNA similarly can be used for the amplification analysis.During the RNA that use to reclaim, can test example such as RNA on insertion, disappearance, displacement, repetition, inversion or the splice variant sudden changes such as (isoforms) of base.In addition, can also detect the rna expression amount.Especially preferably carry out the expression analysis of mRNA, the mutation analysis of K-ras gene and the methylation analysis of DNA etc.In addition, these analyses can be undertaken by known method in this field.In addition, also can use commercially available assay kits such as K-ras gene mutation analysis test kit, methylation detection kit.
The present invention can easier and promptly modulate the ight soil of gathering by feces collection is removed with in the feces collection container of solution S to containing in advance.In addition, having of the application of the invention removed with solution S and the feces collection test kit that contains the feces collection container of removing the usefulness solution S, can carry out nucleic acid recovery method of the present invention more easily.In addition, this feces collection is with can also suitably having feces collection rod etc. in the test kit except that removing with the constituent solution S and the feces collection container that contains it.
The form of the feces collection container that uses among the present invention and size etc. are not particularly limited, and can use the known feces collection container that can take in solvent.Easy in order to handle, the lid of preferred feces collection container and the feces collection container of feces collection rod integral.In addition, in order to control the feces collection amount, more preferably the feces collection rod can be gathered the feces collection container of a certain amount of ight soil.As this known feces collection container, disclosed feces collection container etc. in the special fair 6-72837 communique of Japan is for example arranged.
Fig. 2 and Fig. 3 represent the figure of feces collection of the present invention with the embodiment of feces collection container A that can use in the test kit and B respectively.In addition, feces collection of the present invention is not limited to these feces collection containers with the feces collection container that can use in the test kit.
At first the feces collection container to Fig. 2 describes.The feces collection container A of present embodiment has and feces collection rod 3 incorporate lid 2 and container bodys 1, and the inside of container body 1 is contained to remove and used solution S.The front end of feces collection rod 3 has the cup 3a that can gather a certain amount of ight soil, is furnished with net as sieve in the bottom of this glass 3a.On the other hand, the bottom of container body 1 have with the cup 3a shape protrusion 1a roughly the same and that overlap with the shape of cup 3a.By cup 3a is fitted among the protrusion 1a, make the ight soil gathered among the cup 3a from the net of the bottom cloth of cup 3a, to extrude, therefore ight soil promptly can be scattered in and remove with in the solution S.
The described feces collection container of Fig. 3 has feces collection rod 13 incorporate lid 12 and the container bodys 11 sharp-pointed with front end, is that container body 11 inside have the feces collection container B that contains the sealing bag 15 of removing the usefulness solution S.Vacate the hole 13a that can gather a certain amount of ight soil E in the front of feces collection rod 13.In addition, the movable cover 13b as the lid of hole 13a is connected the both sides of feces collection rod 13 by sliding on feces collection rod 13.Below an embodiment of the using method of this feces collection container B is described.
At first, shown in a of Fig. 3, at first, the movable cover 13b on the feces collection rod 13 is pushed away hole 13a and close lid 12 sides, make hole 13a be in the state of complete opening, feces collection rod 13 is pressed among the ight soil E.So shown in the b of Fig. 3, ight soil E is filled among the 13a of hole.Under this state, cover hole 13a by the front that movable cover 13b is slid into feces collection rod 13, thereby unnecessary ight soil E is separated, therefore can gather the ight soil E (c of Fig. 3) of the volumetric quantity of hole 13a exactly.Then, make movable cover 13b get back to original position and make hole 13a be in the state (d of Fig. 3) of complete opening, will cover 12 then and take in to container body 11 (e of Fig. 3).When feces collection rod 13 is contained to container body 11, the sharp-pointed front end of feces collection rod 13 punctures and contains the bag of removing with solution S 15, thereby container body 11 is removed with solution S and is filled to water level more than the hole 13a that contains ight soil E, and ight soil E directly contacts (f of Fig. 3) with removing with solution S.At this moment, container body 11 tegmentums 12 seal, and therefore remove with solution S can not spill.Then, wait and mobile containers body 11 by carrying, thereby removing with solution S of container 11 inside mixed with ight soil E.Such feces collection container B is owing to solution after inserting feces collection rod 13 in the containers just begins to fill up in the container, even therefore use harmful removing when using solution S as methyl alcohol, also can avoid because of solution spills the accident that causes, in the family also can safe handling.
Embodiment
Then, embodiment is shown the present invention is illustrated in further detail, but the present invention is not limited to following embodiment.In addition, during not special the record, " % " means " volume % ".In addition, MKN45 cell and SW1116 cell use by the ordinary method cultured cells.
[embodiment 1]
Mensuration is present in the bile acide amount in the RNA solution that reclaims by nucleic acid recovery method of the present invention from ight soil.
Respectively getting 1g respectively picks up from the polypropylene tube of 1 healthy people's ight soil to 7 15mL.Divide get after, do not add under the alcoholic acid situation rapidly to wherein 1 carry out RNA rapidly and extract operation.Particularly, add and also to mix phenol mixture " Trizol " (Invitorogen corporate system), add then and mixes chloroform, carry out the centrifugation processing.Separate supernatant (water layer) by this centrifugation processing.In this isolating supernatant, add sodium-acetate and ethanol and stirring.Then, this solution is carried out centrifugation handle, removing supernatant is ethanol, obtains precipitation.This is deposited in room temperature leeward dry doubling is dissolved in the DEPC treated water, obtain the RNA solution (1A) of 100 μ L.
After branch is got, rapidly 6 remaining ight soil are added 99.5% ethanolic soln (remove and use solution S) of 10mL, under 20 ℃, leave standstill 12 minutes (1B), 1 hour (1C), 12 hours (1D), 24 hours (1E), 72 hours (1F), 168 hours (1G) then and preserve these ight soil samples.Through after each shelf time, carry out centrifugally at once, removing supernatant is ethanol (remove and use solution S), obtains the solids component that ight soil is originated.Then, the solids component that the ight soil that obtains is originated extracts operation with (1A) similarly carrying out RNA, obtains the RNA solution of each 100 μ L.
In each the RNA solution that obtains, add margaric acid (C17:0) and 23 nor-deoxycholic acids (23N-DCA) as interior mark, extract with ether.Then, make butyl ester by carboxybutylization to bile acide.In addition, make acetic ester, make the butyl acetyl derivative by glycoloylization to bile acide.Use OV-1701 (GLsci ence corporate system) to utilize GCMS-QP5050 system (Shimadzu Seisakusho Ltd.'s system) that the butyl acetyl derivative that generates is carried out gas chromatographic analysis, measure residual bile acide amount in the RNA solution.
The figure of the bile acide amount in the RNA solution that Fig. 4 is extracted for expression measurement result resulting per 100 μ L.By these results as can be known, by the ight soil sample is soaked (preservation) more than 1 hour in removing the usefulness solution S, can fully remove bile acide as inhibitory substance A.And then, between 1 hour to 24 hours, can remove bile acide sharp.In addition, the ight soil sample surpasses 72 hours sample with the soak time in the solution S and almost can't see the variation of removing efficient removing.
[embodiment 2]
Affirmation is suppressed by the reaction of the RT-PCR that bile acide causes.Particularly, with the RNA that reclaimed from culturing cell MKN45 cell as template, the main component of adding bile acide in the reaction solution of real-time (One-Step real time) RT-P CR of the step of amplification GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA is the dilution series of Sodium desoxycholate, confirms the rejection condition that is caused by Sodium desoxycholate.
Each reagent is mixed according to the specification sheets limit of single stage method Prime Script RT-PCR test kit (TaKaRa) in the limit, in reaction solution, add Sodium desoxycholate, the ultimate density that makes Sodium desoxycholate is 0,1,2,4,10,15,20 μ g/25 μ L, carries out 42 ℃ of 5 minutes, the reverse transcription reaction in 95 ℃ of 10 second.Then, will repeat 40 circulations by 95 ℃ of 5 second, the reaction conditions that constitutes second for 60 ℃ 30 and carry out PCR.At this moment, use TaqMan gene expression analysis (Gene Expression Assays), stock's (Inventoried) GAPDH to detect with TaqMan primer and probe.
Analyze synthetic reagent by ABI Prism 7700 sequence detection systems (S equence Detection System) (Applied Biosystems corporate system), confirm the amplification efficiency of TaqMan PCR.Calculate copy number by the CT value shown in the plasmid DNA of concentration known, the evaluation that suppresses by the reduction degree of the guess value of the initial stage nucleic acid amount that obtains thus.
Fig. 5 is that transverse axis, GAPDH amplification amount are the figure of the measurement result of the longitudinal axis for expression with the Septochol na concn.Can be confirmed by these results, the concentration of the Sodium desoxycholate that contains when reaction system increases to 4 μ g/25 μ L when above, can confirm the inhibition of the nucleic acid amplification reaction that inhibitory substance A caused.
At this, be template with 5 μ L resulting RNA under the condition of embodiment 1, making reaction system is that 25 μ L carry out RT-PCR.At this moment, when to use the shelf time be 1 hour RNA (1C), the bile acide concentration in the reaction solution was about 3.75 μ g/25 μ L, and when to use the shelf time be 12 hours RNA (1D), the bile acide concentration in the reaction solution was about 1.6 μ g/25 μ L.That is, understood, as long as the shelf time is more than 1 hour, the influence that bile acide caused of bringing into from ight soil in the time of can suppressing to reclaim nucleic acid effectively to nucleic acid amplification reaction.In addition, consider also that other impurity things are also influential under the situation of the nucleic acid that reclaims from ight soil, therefore think also can produce inhibition than the also little bile acide amount of concentration (about 1.6 μ g/25 μ L) of preservation in the time of 12 hours.Therefore as can be known, more preferably preserve more than 12 hours.
[embodiment 3]
Respectively getting 1g respectively picks up from the polypropylene tube of 1 healthy people's ight soil to 3 15mL.Divide get after, to 1 70% ethanolic soln (remove and use solution S) that adds 10mL wherein, under room temperature (20 ℃), left standstill 1 minute then, rapidly as ight soil sample (3A).Branch is got the back fully disperses ight soil to other 1 70% ethanolic soln (remove and use solution S) that adds 10mL, at room temperature leaves standstill then and preserves 18 hours, as ight soil sample (3B).Divide and to get the back and ight soil is fully disperseed, at room temperature left standstill then and preserve 36 hours 1 remaining 70% ethanolic soln (remove and use solution S) that adds 10mL, with it as ight soil sample (3C).Through after each shelf time, immediately each sample is carried out centrifugally, removing supernatant is ethanol (remove and use solution S), obtains the solids component that ight soil is originated.
In addition, from removed each ight soil sample of alcoholic acid, reclaim RNA at any time.Particularly, in the solids component in each ight soil source, add and mix phenol mixture " Trizol " (Invitorogen corporate system), add then and mixes chloroform, carry out the centrifugation processing.Obtain supernatant (water layer) by this centrifugation processing.In this supernatant, add sodium-acetate and ethanol, carry out centrifugation after the stirring and handle.Obtain precipitation, air-dry then this throw out by this centrifugation processing.These throw outs are dissolved in the DEP C treated water, obtain RNA solution.
Use the part (5 μ L) in each RNA solution, adopt as reverse transcription reaction and synthesize cDNA with the ReverTra Ace qP CR RT Kit of test kit.With this cDNA is template, to the 2 * TaqMan PCR master mix that wherein adds 12.5 μ L (Perkin-Elmer Applied Biosystems corporate system), and add people GAPDH respectively and make with reverse primer (sequence number 2:5 '-GAAGATGGTGATGGGATTTC-3 ') with forward primer (sequence number 1:5 '-GAAGGTGAAGGTCGGAGTC-3 ') and people GAPDH that ultimate density is 900nmol in the reaction solution, final volume is 25 μ L, modulation PCR solution.Utilize ABI Prism 7700 (Perkin-Elmer Applied Biosystems corporate system) this PCR solution to be used the pcr analysis of SYBR green.The thermal cycling of PCR is repeating 95 ℃ of 30 second, 55 ℃ of 30 second, 72 ℃ of 30 second to carry out under 45 round-robin conditions after 95 ℃ of 10 minutes sex change circulations.Quantitatively the result based on the fluorescence intensity that obtains as template with the dilution series of utilizing the known standard plasmid of concentration carries out.
When the RNA in sample (3A) source was used for template, (3B) compared with sample, can see the reduction of about amplification efficiency more than 80%.In addition, the difference of sample (3B) and sample (3C) is about about 10%, does not see the poor of sample (1A) and the sort of degree of sample (1B).
That is, by these results as can be known, use to add in ight soil and remove the nucleic acid that obtains with the nucleic acid recovery method of the present invention of solution S, immersion and preservation certain hour, the amplification efficiency of nucleic acid is fine.This is because by nucleic acid recovery method of the present invention, can reduce the amount of the inhibitory substance A in the ight soil source in the nucleic acid of recovery.In addition, in the present embodiment,,, infer therefore that bile acide contained in ight soil inhibitory substance A in addition also is dissolved to remove because the amplification efficiency of the nucleic acid that reclaims is good though do not have to confirm by removing the material of removing with the solution S stripping what is.
[embodiment 4]
Respectively getting 1g respectively picks up from the polypropylene tube of 1 healthy people's ight soil to 3 15mL.Divide get after, to 1 70% ethanolic soln that adds 10mL wherein, under 4 ℃, left standstill 1 minute then, rapidly as ight soil sample (4A).Branch is got the back fully disperses ight soil to other 1 70% ethanolic soln (remove and use solution S) that adds 10mL, leaves standstill and preserves under 4 ℃ 12 hours then, as ight soil sample (4B).Divide and to get the back and ight soil is fully disperseed, under 4 ℃, left standstill and preserve 72 hours then 1 remaining 70% ethanolic soln (remove and use solution S) that adds 10mL, with it as ight soil sample (4C).Through after each time, remove ethanol by centrifugation at any time,, be PBS by the centrifugal supernatant of removing then then with the PBS washing of 7mL.
Extract RNA similarly to Example 3 and carry out reverse transcription reaction.Then, the 16S rRNA gene by utilizing bacterium detects colibacillary gene with the 16S rRNA gene of forward primer (sequence number 3:5 '-AGGAGGTGATCCAACCGCA-3 ') and bacterium with the PCR in real time of reverse primer (sequence number 4:5 '-AACTGGAGGAAGGTGGGGAT-3 ').When the RNA in sample (4A) source was used for template, (4B) compared with sample, can see the reduction of about amplification efficiency more than 70%.In addition, the difference of sample (4B) and sample (4C) is about about 30%.
That is, by these results as can be known, even when the temperature when preserving is 4 ℃, use the resulting nucleic acid of nucleic acid recovery method of the present invention, the amplification efficiency of nucleic acid is also fine,, infers with the bile salt to be that the inhibitory substance A of representative is removed from the nucleic acid that reclaims that is.
[embodiment 5]
Replace the ethanol that uses among the embodiment 1, make the alcoholic solution that has added 55% methyl alcohol, 5% Virahol.This alcoholic solution is used solution S as removing, prepare alcoholic solution and soak faecal samples, make the sample that has changed immersion (preservation) time similarly to Example 1.That is, divide get after, to 1 alcoholic solution that adds 10mL wherein ight soil is fully disperseed rapidly, under room temperature (20 ℃), left standstill 1 minute then, as ight soil sample (5A).Branch is got the back fully disperses ight soil to other 1 alcoholic solution that adds 10mL, at room temperature leaves standstill then and preserves 12 hours, as ight soil sample (5B).Divide and to get the back and ight soil is fully disperseed, at room temperature left standstill then and preserve 36 hours 1 remaining alcoholic solution that adds 10mL, with it as ight soil sample (5C).At any time from these samples, remove alcoholic solution by centrifugation, obtain the solids component in ight soil source.Then, with the solids component that the 99.5% washing with alcohol ight soil of 7mL is originated, by centrifugal i.e. 99.5% ethanol of supernatant of removing, the solids component that ight soil is originated carries out air-dry.Then, with " Trizol " and chloroform extraction RNA.The water layer that obtains is added ethanol, stir well with vortex mixer, in reclaiming with post (silicagel column), adds the RNA of RNeasy midi kit (Qiagen corporate system) this water layer and alcoholic acid mixed solution and centrifugal then, according to appended working specification, this RNA recovery is carried out washing operation and RNA stripping operation with post, thereby reclaim RNA.
Use the part in each the RNA solution that is reclaimed, used the pcr analysis of SYBR green similarly to Example 3.Its result, when the RNA in sample (5A) source was used for template, (5B) compared with sample, can see the reduction of about 40% amplification efficiency.In addition, the difference of sample (5B) and sample (5C) is about about 20%, for the pact of the difference of sample (5A) and sample (5B) half.
That is, by these results as can be known,, also can extract inhibitory substance A such as removing bile salt and reclaim highly purified nucleic acid even use the alcohol conduct beyond the ethanol to remove when using solution S.In addition, temporarily use organic solvent extraction RNA after, further carry out purifying, thereby remove materials such as carbohydrate and nucleic acid resolvent with silicagel column.Thus, the amplification efficiency of the nucleic acid in the ight soil rises.
[embodiment 6]
In the healthy people's of 4g ight soil, mix 5.0 * 10
5Individual K-ras gene codon 12 sports the culturing cell SW1116 cell in the human colon carcinoma source of GCT, with this material that mixes as the doubtful ight soil of colorectal carcinoma patient.Respectively get in the polypropylene tube of this ight soil to 6 of 0.5g 15mL respectively.Divide get after, to 2 modification ethanol (alcoholic solution of 55% ethanol and 5% Virahol) that add 10mL wherein ight soil is fully disperseed rapidly, under room temperature (20 ℃), left standstill 1 minute then, as ight soil sample (6A).Divide after getting to 2 other modification ethanol (remove and use solution S) that add 10mL ight soil fully to be disperseed rapidly, at room temperature left standstill then and preserve 12 hours, as ight soil sample (6B).Divide and to get the back and to 2 remaining modification ethanol (remove and use solution S) that add 10mL ight soil is fully disperseed rapidly, at room temperature left standstill then and preserve 36 hours, with it as ight soil sample (6C).
(6A), each sample of (6B), (6C) is through after each shelf time, removes modification ethanol by centrifugation at any time, obtains the solids component in ight soil source.Then, with the solids component that the 99.5% washing with alcohol ight soil of 7mL is originated,, that this solids component is air-dry by centrifugal i.e. 99.5% ethanol of supernatant of removing.
In addition, from the solids component of having removed each ight soil source of alcoholic acid, reclaim dna solution at any time.That is, use ight soil source DNA extraction test kit " QIAamp DNA Stool Mini Kit " (Qiagen corporate system) from the solids component in each ight soil source, to reclaim DNA.With absorbance method the concentration of the DNA of recovery is carried out quantitatively, the result can reclaim the almost DNA of isodose from the solids component in each ight soil source.
Use the DNA that reclaims, detect the GCT mutant nucleotide sequence of K-ras codon 12 with allele-specific PCR (allele specific-PCR) method.That is, so that the 3 ' terminal mode that is complementary with the GCT mutant nucleotide sequence of the primer of positive-sense strand side designs, thus amplification GCT mutant allele.In addition, the primer of antisense strand side derives from the base sequence of intron part.Average each reaction template nucleic acid with TaKaRa Ex Taq (TaKaRa corporate system), 1 * Ex Taq Buffer, 200 μ M dNTP Mixture and the 10ng corresponding forward primer of K-ras codon 12GCT mutant codon (sequence number 5:5 '-ACTTGTGGTAGTTGGAGCTGC-3 ') and reverse primer (sequence number 6:5 '-CTCATGAAAATGGTCAGAGAAACC-3 '), 1.25U that use each 50pmol carries out PCR.Temperature condition be 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, carry out 35 circulations.
After the PCR reaction, confirm amplified production by the agarose gel electrophoresis made from 3%NuSieve (FMC Corp.'s system), results verification is to the amplified production of 179bp.Its result, when the DNA in sample (6A) source was used for template, one of them can't see amplification 2 samples.In addition, the band of the another one also DNA than sample (6B) source is light a lot, can see the reduction of amplification efficiency.In addition, the DNA in the DNA in 2 samples (6B) source and 2 samples (6C) source increases well, can not see the difference of band concentration substantially.
Can infer by above result, the shelf time aspect, the time, the amplification efficiency of longer nucleic acid was good more more, that is, suppress nucleic acid amplification bile salt etc. to remove efficient good more.
As can be known, by using the DNA that is reclaimed by nucleic acid recovery method of the present invention, thereby even transgenations etc. are had relatively high expectations the foranalysis of nucleic acids of accuracy, also can carry out foranalysis of nucleic acids accurately.In addition, use in the present embodiment and mixed Virahol and alcoholic acid modification ethanol is used solution S as removing, but equal result can access with 50% the ethanolic soln that uses identical determining alcohol the time.
[embodiment 7]
Use feces collection container A modulation ight soil sample as shown in Figure 2 and reclaim nucleic acid.This feces collection container A has and feces collection rod 3 incorporate lid 2 and container bodys 1, and the feces collection container A with 59% ethanolic soln of solution S is removed in the conduct that contains 5mL for inside.In addition, the front end of feces collection rod 3 possesses and has 4 diameters the cup 3a of the 0.5mL capacity in the hole that is 3mm.
At first, use feces collection rod 3 to gather about 0.5g ight soil to glass 3a, and be inserted among this feces collection container A and close the lid 2.So the protrusion 1a of the bottom of container body 1 extrudes ight soil from the hole of cup 3a, thread ight soil is scattered in the solution.With synthetic like this ight soil sample as ight soil sample (7A).
On the other hand, similarly gather about 0.5g ight soil to the 15mL polypropylene tube of 59% ethanolic soln that contains 5mL with ight soil sample (7A), making the ight soil and the volume ratio of removing with solution S is 1: 10, with its sample (7B) in contrast.
Ight soil sample (7A) and control sample (7B) were preserved 2 days down at 30 ℃, reclaimed RNA then similarly to Example 2.At this moment,, compare, show darker dark brown with the ethanol of removing from control sample (7B) from the ethanol that ight soil sample (7A) is removed.In addition, reclaim RNA, synthetic cDNA similarly to Example 3 after, carry out the quantitative PCR of people GAPDH gene.When the RNA in sample (7B) source is used for template, compare the reduction that to see about 40% amplification efficiency with the RNA in sample (7A) source.
Its result can infer, because use feces collection container A as shown in Figure 2 removes with in the solution S thereby can promptly ight soil be scattered in, more high purity ground reclaims nucleic acid.In addition, by using this feces collection container A, thereby the feces collection person can be easy after gathering ight soil and promptly carry out the modulation and the preservation of ight soil sample, therefore can reduce the part of the staff cost of checking process operations person.
[embodiment 8]
Use feces collection container B modulation ight soil sample as shown in Figure 3 and reclaim nucleic acid.Feces collection rod 13 front ends of this feces collection container are sharp-pointed, have the hole 13a of 2.5mL capacity.In addition, feces collection container B has and feces collection rod 13 incorporate lid 12 and container bodys 11, has the sealing bag 15 that contains as removing with 59% methanol solution of solution S in container body 11 inside.
At first, use bag 15 to contain the feces collection container B of 59% methanol solution of 5mL, according to the ight soil of the about 0.1g of acquisition order of Fig. 3,59% methanol solution that makes the interior 5mL of bag 15 mixes with ight soil and modulates ight soil sample (8A).This ight soil sample (8A) was left standstill and preserves under 18 ℃ 36 hours, reclaim RNA then similarly to Example 5, used the pcr analysis of SYBR green, results verification is good to amplification efficiency.
Also clear by this result, carry out nucleic acid recovery method of the present invention by using feces collection container B as shown in Figure 3, can reclaim nucleic acid in high purity ground, therefore can seek feces collection with lightweight, the miniaturization of test kit with guarantee security.
[embodiment 9]
Use ight soil apparatus for samples treatment as shown in Figure 1 carries out the operation (B) in the nucleic acid recovery method of the present invention automatically, reclaims nucleic acid from the ight soil sample.
At first, similarly to Example 7, modulate 2 pipes have mixed ight soil and 59% ethanolic soln (remove and use solution S) in feces collection container A shown in Figure 2 ight soil sample.Just after sample is at room temperature preserved 12 hours with this 2 night soil management, open the cover of feces collection container, be fixed in the centrifuge mechanism in the ight soil apparatus for samples treatment.Fixed ight soil sample is separated into the solids component that supernatant partly is ethanolic soln (remove and use solution S) and ight soil source by centrifugal separation process.Aspirate the just supernatant part of sample and discarded to the waste liquid recoverer of 1 night soil management with solution suction discharge nozzle.After discarded, use the scrubber layer Washing spray nozzle, remaining ight soil sample is similarly carried out removing of supernatant.
After removing supernatant, can from the ight soil solids component that obtains, reclaim RNA similarly to Example 5.
[embodiment 10]
Respectively getting 1g respectively picks up from the polypropylene tube of 1 healthy people's ight soil to 8 15mL.After branch is got, 70% ethanolic soln (remove and use solution S) that adds 6mL disperses ight soil fully and modulates the ight soil sample rapidly, under-4 ℃ (10A), 0 ℃ (10B), 4 ℃ (10C), 8 ℃ (10D), 12 ℃ (10E), 16 ℃ (10F), 20 ℃ (10G), 24 ℃ of (10H) each conditions synthetic ight soil sample is left standstill and preserves 24 hours then.
Each sample is through after each shelf time, and is centrifugal and to remove supernatant be ethanolic soln, obtains the solids component in ight soil source.
Then, from the solids component that the ight soil that obtains is originated, reclaim RNA similarly to Example 1.Use the part in each the RNA solution that is reclaimed, used the pcr analysis of SYBR green similarly to Example 3.
To be shown in table 1 with the analytical results of reacted each the ight soil sample (sample) of SYBR green.Respectively with sample (10A) and (10B) RNA in source do not confirm amplification when being used for template.On the other hand, when the RNA that sample (10C)～sample (10F) is originated is used for template respectively, confirm amplification.In addition, under the situation of the RNA in sample (10G) and sample (10H) source, can see the amplification about the several times of RNA in sample (10C)～sample (10F) source.
That is, storage temperature is more than 4 ℃ the time, inhibitory substance A to remove efficient fine, can remove inhibitory substance and nucleic acid amplification efficient in the time of more than 20 ℃ better and rise.
[table 1]
| The ight soil sample | 10A | 10B | 10C | 10D | 10E | 10F | 10G | 10H |
| Storage temperature | -4 |
0 |
4 |
8 |
12 |
16 |
20℃ | 24℃ |
| The nucleic acid amplification amount | - | - | + | + | + | + | ++ | ++ |
[embodiment 11]
Respectively getting 1g respectively picks up from the polypropylene tube of 1 healthy people's ight soil to 9 15mL.After branch is got, 70% ethanolic soln (remove and use solution S) that adds rapidly 6mL fully disperses ight soil and modulates the ight soil sample, leaves standstill and preserves under the shelf time is each condition of 1 minute (11A), 10 minutes (11B), 1 hour (11C), 12 hours (11D), 24 hours (11E), 36 hours (11F), 48 hours (11G), 72 hours (11H), 168 hours (11I) then.In addition, storage temperature is 20 ℃ of certain temperatures.
Each sample reclaims RNA similarly to Example 10 through after each shelf time, analyzes with quantitative PCR then.Analytical results is shown in table 2.
Respectively with sample (11A) and (11B) RNA in source do not confirm amplification when being used for template, but when the RNA in sample (11C) source is used for template, confirm amplification.In addition, under the situation of the RNA in sample (11D) source, can see the amplification about the several times of RNA in sample (11C) source.Under the situation of the RNA that sample (11E), (11F), (11G) originate, can see about 3 times the amplification of the RNA in sample (11C) source, sample (11H) and the RNA that (11I) originates can see about 1.5 times the amplification of the RNA in sample (11D) source.
That is, the shelf time is more than 1 hour the time, inhibitory substance A to remove efficient fine, can remove inhibitory substance better in the time of more than 24 hours, can further remove in the time of more than 72 hours, do not see the decline of nucleic acid amplification efficient in the time of 168 hours yet.
[table 2]
| The ight soil sample | ?11A | 11B | 11C | 11D | 11E | 11F | 11G | 11H | ?11I |
| Shelf time | 1 |
10 minutes | 1 |
12 hours | 24 hours | 36 hours | 48 hours | 72 hours | 168 hours |
| The nucleic acid amplification amount | ?- | - | + | ++ | +++ | +++ | +++ | ++++ | ?++++ |
[reference example 1]
Respectively getting 1g respectively picks up from the polypropylene tube of 1 healthy people's ight soil to 3 15mL.Divide get after, use liquid nitrogen that wherein 1 is carried out freezing treatment rapidly, as ight soil sample (1).After branch is got, ight soil is fully disperseed, at room temperature left standstill then 1 hour, as ight soil sample (2) to other 1 70% ethanolic soln that adds 10mL.Divide get after, remaining 1 is transferred in the abstraction process under not adding the condition of solution etc., with it as ight soil sample (3).
Then, from each ight soil sample, reclaim RNA.Particularly, in each ight soil sample, add the phenol mixture " Trizol " (Invitorogen corporate system) of 3mL, use the homogenizer thorough mixing more than 30 seconds.Add the 3mL chloroform then, use the vortex mixer thorough mixing, then with 12,000 * g, under 4 ℃, carry out centrifugation in 20 minutes and handle.To handle the supernatant (water layer) that obtains by this centrifugation and feed the RNA recovery of RNeasy midi kit (Qiagen corporate system) with in the post, according to appended working specification, this RNA recovery is carried out washing operation and RNA stripping operation with post, thereby reclaim RNA.Use NanoDrop (NanoDrop corporate system) that the RNA that reclaims is carried out quantitatively.
Be less than the RNA amount that reclaims the ight soil sample (1) that after gathering, has carried out freezing treatment rapidly a little from using as of the present invention removing with the RNA amount that reclaims the synthetic ight soil sample of the ethanolic soln of solution S (2), but compare with gathering the ight soil sample (3) that has carried out nucleic acid extraction afterwards rapidly, can reclaim very many RNA.By these results as can be known, removing with solution S of the application of the invention modulated, even be modulation at room temperature, also can obtain to reclaim very effectively the ight soil sample of nucleic acid.Under such situations such as examination, when the patient carries out feces collection the own home, be desirably near carry out the ight soil sample room temperature modulation, and of the present invention removing with solution S can fully be satisfied such requirement.
[reference example 2]
Divide separately and get 1g and pick up from the polypropylene tube of 1 healthy people's ight soil to 2 15mL.Divide get after, to 1 70% ethanolic soln that adds 10mL wherein ight soil is fully disperseed, at room temperature left standstill then 1 hour, as ight soil sample (4).Remove the ethanol in the ight soil sample (4) then, then extract operation.Divide get the ight soil sample after, in remaining 1 phosphoric acid buffer (pH7.0) that is suspended in 10mL,, remove the residue also bigger than cell by the slight centrifugal supernatant that reclaims.The supernatant that reclaims centrifugal 15 minutes with 3000rpm reclaims as sedimentary cell.Add 70% ethanol of 9mL in precipitation, mixed back centrifugal 10 minutes with 3000rpm as ight soil sample (5), is then extracted operation with throw out.Particularly, in each ight soil sample, add the phenol mixture " Trizol " (Invitorogen corporate system) of 3mL, use the homogenizer thorough mixing more than 30 seconds.Then, add the 3mL chloroform, use the vortex mixer thorough mixing, then with 12,000 * g, under 4 ℃, carry out centrifugation in 20 minutes and handle.The supernatant (water layer) that will be obtained by this centrifugation processing feeds the RNA recovery of RNeasy midi kit (Qiagen corporate system) and uses post, according to appended working specification, this RNA recovery is carried out washing operation and RNA stripping operation with post, thus recovery RNA.Use NanoDrop (NanoDrop corporate system) that the RNA that reclaims is carried out quantitatively.
Use the part (5 μ L) of each RNA solution, use as reverse transcription reaction and synthesize cDNA with the ReverTra Ace qPCR RT Kit of test kit.With this cDNA is template, to the 2 * TaqMan PCR master mix that wherein adds 12.5 μ L (Perkin-Elmer Applied Biosystems corporate system), and add people GAPDH respectively and make with reverse primer (sequence number 2:5 '-GAAGATGGTGATGGGATTTC-3 ') with forward primer (sequence number 1:5 '-GAAGGTGAAGGTCGGAGTC-3 ') and people GAPDH that ultimate density is 900nmol in the reaction solution, final volume is 25 μ L, modulation PCR solution.Utilize ABI Prism 7700 (Perkin-Elmer Applied Biosystems corporate system) to use SYBR green that this PCR solution is carried out pcr analysis.The thermal cycling of PCR is repeating 95 ℃ of 30 second, 55 ℃ of 30 second, 72 ℃ of 30 second to carry out under 45 round-robin conditions after 95 ℃ of 10 minutes sex change circulations.Quantitatively the result based on the fluorescence intensity that obtains as template with the dilution series of utilizing the known standard plasmid of concentration carries out.
When the RNA in sample (5) source is used for template, compare, can see the reduction of about amplification efficiency more than 50% with sample (4).
Promptly, by these results as can be known, use the ight soil of gathering not made suspension but to be soaked in immediately and remove with in the solution S and preserve the nucleic acid recovery method of the present invention of certain hour and the nucleic acid that obtains, compare with the temporary transient nucleic acid of ight soil being made the nucleic acid recovery method of suspension and obtaining of use, inhibitory substance A is removed from the nucleic acid that reclaims, and the recovery loss is less, and the amplification efficiency of nucleic acid is fine.
In addition, the PCR method of archaeal dna polymerase is used in the nucleic acid amplification reaction utilization in the foregoing description and the reference example, but can comprise that also the nucleic acid amplification of the reverse transcription reaction that has made up the use RNA polymerase is RT-PCR method, NASBA method and TRC method etc.
Utilizability on the industry
Utilize process for recovering nucleic acid of the present invention can effectively remove nucleic acid amplification reaction contained in the ight soil and suppress material, from the ight soil sample, effectively reclaim highly purified nucleic acid, therefore can be used in especially in the fields such as clinical examination such as using having regular physical checkups of ight soil sample.
Claims (39)
1. a method that reclaims nucleic acid from the ight soil sample is characterized in that, it is the method that high purity ground reclaims nucleic acid from ight soil, has following operation:
(A) ight soil of gathering being added to the water-miscible organic solvent is that the nucleic acid amplification reaction inhibitory substance of effective constituent is removed with in the solution and modulated the ight soil sample, and above-mentioned ight soil sample preserved specific time, thereby make the operation of nucleic acid amplification reaction inhibitory substance stripping;
(B) afterwards, removing the nucleic acid amplification reaction inhibitory substance from the ight soil sample removes with solution and reclaims the operation of the solids component in ight soil source in operation (A); With
(C) operation of recovery nucleic acid from the solids component that the ight soil that reclaims operation (B) is originated.
2. the method that reclaims nucleic acid from the ight soil sample according to claim 1 wherein, is not made suspension with the ight soil of gathering, and removes with in the solution modulation ight soil sample but add the ight soil of gathering to the nucleic acid amplification reaction inhibitory substance immediately.
3. according to claim 1 or the described method that reclaims nucleic acid from the ight soil sample of claim 2, wherein, above-mentioned water-miscible organic solvent is water-soluble alcohol and/or ketone.
4. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～3, wherein, the concentration of above-mentioned water-miscible organic solvent is more than 30%.
5. according to claim 3 or the 4 described methods that from the ight soil sample, reclaim nucleic acid, wherein, above-mentioned water-miscible organic solvent contain be selected from the group of forming by ethanol, propyl alcohol and methyl alcohol more than a kind as water-soluble alcohol.
6. the method that reclaims nucleic acid from the ight soil sample according to claim 1, wherein, above-mentioned water-miscible organic solvent is an ethanol.
7. according to claim 3 or the 4 described methods that reclaim nucleic acid from the ight soil sample, wherein, above-mentioned water-miscible organic solvent contains acetone and/or methylethylketone as ketone.
8. according to each described method that from the ight soil sample, reclaims nucleic acid in the claim 1～7, wherein, remove the mixture ratio of using solution about above-mentioned ight soil and above-mentioned nucleic acid amplification reaction inhibitory substance, with respect to faecal volume 1, it is more than 1 that the nucleic acid amplification reaction inhibitory substance is removed with liquor capacity.
9. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～8, wherein, the nucleic acid amplification reaction inhibitory substance of removing from the ight soil sample in the above-mentioned operation (B) is removed with solution and is undertaken by centrifugal separation.
10. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～9, wherein, in the above-mentioned operation (A), the time of preserving the ight soil sample is more than 1 hour.
11. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～9, wherein, in the above-mentioned operation (A), the time of preserving the ight soil sample is more than 12 hours.
12. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～9, wherein, in the above-mentioned operation (A), the time of preserving the ight soil sample is more than 24 hours.
13. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～9, wherein, in the above-mentioned operation (A), the time of preserving the ight soil sample is more than 72 hours.
14. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～13, wherein, in the above-mentioned operation (A), the temperature of preserving the ight soil sample is more than 4 ℃.
15. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～13, wherein, in the above-mentioned operation (A), the temperature of preserving the ight soil sample is more than 20 ℃.
16. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～15, wherein, above-mentioned nucleic acid amplification reaction inhibitory substance is removed with solution and is contained tensio-active agent.
17. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～16, wherein, above-mentioned nucleic acid amplification reaction inhibitory substance is removed with solution and is contained tinting material.
18. according to each described method that from the ight soil sample, reclaims nucleic acid in the claim 1～17, wherein, above-mentioned operation (C) is following operation: recovery simultaneously often occupies the nucleic acid in enterobacteria source and the nucleic acid of the biogenetic derivation beyond the Chang Ju enterobacteria from the solids component that the ight soil that reclaims operation (B) is originated.
19. the method that reclaims nucleic acid from the ight soil sample according to claim 18, wherein, the above-mentioned biology that often occupies beyond the enterobacteria is a mammalian cell.
20. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 1～19, wherein, above-mentioned operation (C) has following operation:
(a) make protein denaturation in the solids component in the ight soil source of reclaiming in the operation (B), and make the operation that often occupy in biology enterobacteria and Chang Ju enterobacteria beyond stripping of nucleic acid from the solids component in above-mentioned ight soil source; With
(b) reclaim the operation of the nucleic acid of stripping in the above-mentioned operation (a).
21. the method that from the ight soil sample, reclaims nucleic acid according to claim 20, wherein, above-mentioned operation (a) afterwards, above-mentioned operation (b) has (c) before and removes the proteinic operation of sex change by above-mentioned operation (a).
22. according to claim 20 or the 21 described methods that from the ight soil sample, reclaim nucleic acid, wherein, in the above-mentioned operation (a) proteinic sex change be to use a kind of being selected from the group of forming by chaotropic salt, organic solvent and tensio-active agent with on carry out.
23. the method that reclaims nucleic acid from the ight soil sample according to claim 22, wherein, above-mentioned organic solvent is a phenols.
24. according to each described method that reclaims nucleic acid from the ight soil sample in the claim 21～23, wherein, proteinic the removing of the sex change in the above-mentioned operation (c) is to use chloroform to carry out.
25., it is characterized in that the recovery of the nucleic acid in the above-mentioned operation (b) has following operation according to each described method that from the ight soil sample, reclaims nucleic acid in the claim 20～24:
(b1) make the nucleic acid of stripping in the above-mentioned operation (a) be adsorbed in the operation of inorganic supporting body; With
(b2) make the operation of the nucleic acid of absorption in the above-mentioned operation (b1) from the inorganic supporting body stripping.
26. a nucleic acid, its nucleic acid for reclaiming by each described method that from the ight soil sample, reclaims nucleic acid in the claim 1～25.
27. a method for nucleic acid analysis, it uses the nucleic acid that reclaims that the nucleic acid in mammalian cell source is analyzed, and the nucleic acid of this recovery is to utilize the nucleic acid that each described nucleic acid recovery method reclaims from the ight soil sample in the claim 1～26.
28. method for nucleic acid analysis according to claim 27, wherein, above-mentioned mammalian cell is the digestive tube cell.
29. method for nucleic acid analysis according to claim 27, wherein, the cell of above-mentioned mammalian cell for coming off from large intestine.
30. according to each described method for nucleic acid analysis in the claim 27～29, wherein, the nucleic acid in above-mentioned mammalian cell source is the marker of indication neoplastic transformation.
31. according to each described method for nucleic acid analysis in the claim 27～29, wherein, the nucleic acid in above-mentioned mammalian cell source is the struvite chylopoietic disease's of indication marker.
32. according to each described method for nucleic acid analysis in the claim 27～31, wherein, above-mentioned analysis be selected from the group of forming by the methylation analysis of the mutation analysis of the expression analysis of mRNA, K-ras gene and DNA more than a kind.
33. ight soil apparatus for samples treatment, it comprises that solution removes mechanism, this solution is removed mechanism and is used for removing following nucleic acid amplification reaction inhibitory substance from the ight soil sample and removes and use solution, and this ight soil sample is that the ight soil that will gather is soaked in the water-miscible organic solvent is that the nucleic acid amplification reaction inhibitory substance of effective constituent is removed with the ight soil sample of preserving in the solution behind the specific time.
34. ight soil apparatus for samples treatment according to claim 33, wherein, above-mentioned solution is removed mechanism and is had centrifuge mechanism, solution suction output mechanism and waste liquid recoverer.
35. ight soil apparatus for samples treatment according to claim 34, wherein, above-mentioned solution suction output mechanism is a solution suction discharge nozzle, and this ight soil apparatus for samples treatment also has the mechanism of the above-mentioned solution suction of washing discharge nozzle.
36. a nucleic acid amplification reaction inhibitory substance is removed and used solution, it is to be used to modulate nucleic acid to reclaim the solution of using the ight soil sample, is effective constituent with the water-miscible organic solvent, in order to reduce the nucleic acid amplification reaction inhibitory substance in the nucleic acid that reclaims.
37. nucleic acid amplification reaction inhibitory substance according to claim 36 is removed and used solution, wherein, above-mentioned water-miscible organic solvent is water-soluble alcohol and/or ketone.
38. remove according to the described nucleic acid amplification reaction inhibitory substance of claim 37 and to use solution, wherein, the concentration of above-mentioned water-miscible organic solvent is more than 30%.
39. a feces collection test kit, it has each described nucleic acid amplification reaction inhibitory substance in the claim 36～38 and removes with solution and contain this nucleic acid amplification reaction inhibitory substance and remove feces collection container with solution.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008189684 | 2008-07-23 | ||
| JP2008-189684 | 2008-07-23 | ||
| PCT/JP2009/063163 WO2010010914A1 (en) | 2008-07-23 | 2009-07-23 | Method for collection of nucleic acid from fecal sample, method for analysis of nucleic acid, and apparatus for treatment of fecal sample |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102105583A true CN102105583A (en) | 2011-06-22 |
Family
ID=41570374
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009801285392A Pending CN102105583A (en) | 2008-07-23 | 2009-07-23 | Method for collection of nucleic acid from fecal sample, method for analysis of nucleic acid, and apparatus for treatment of fecal sample |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US20110183332A1 (en) |
| EP (1) | EP2314677A4 (en) |
| JP (1) | JP5710969B2 (en) |
| CN (1) | CN102105583A (en) |
| WO (1) | WO2010010914A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110291197A (en) * | 2016-10-27 | 2019-09-27 | 基因复制有限公司 | Detection method |
| CN112501157A (en) * | 2020-11-30 | 2021-03-16 | 广东军融科创科技有限公司 | Kit for extracting DNA from human excrement sample, extraction method and application |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103890163B (en) | 2011-06-19 | 2016-09-14 | 阿博根公司 | Devices, solutions and methods for sample collection |
| DE102012015706A1 (en) * | 2012-08-07 | 2014-02-13 | Prionics Ag | Sampling device for DNA samples in particular |
| ES2661684T3 (en) | 2014-03-03 | 2018-04-03 | Fundacio Institut D'investigació Biomèdica De Girona Dr. Josep Trueta | Method to diagnose colorectal cancer from a sample of human feces by quantitative PCR |
| JP6670765B2 (en) | 2014-03-07 | 2020-03-25 | ディーエヌエー ジェノテック インク | Compositions and methods for stabilizing nucleic acids in biological samples |
| AU2019276577B2 (en) | 2018-06-01 | 2025-09-11 | Geneoscopy, Inc. | Detection method |
Family Cites Families (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5998483A (en) * | 1991-09-20 | 1999-12-07 | Camiener; Gerald W. | Glyoxal-containing preservative compositions |
| CA2132874C (en) * | 1992-04-01 | 2003-08-19 | Bert Vogelstein | Methods of detecting mammalian nucleic acids isolated from stool specimen and reagents therefor |
| DE19530132C2 (en) * | 1995-08-16 | 1998-07-16 | Max Planck Gesellschaft | Process for the purification, stabilization or isolation of nucleic acids from biological materials |
| US5670325A (en) * | 1996-08-14 | 1997-09-23 | Exact Laboratories, Inc. | Method for the detection of clonal populations of transformed cells in a genomically heterogeneous cellular sample |
| MXPA97000542A (en) * | 1996-03-25 | 2004-05-27 | Univ Mexico Nacional Autonoma | Specific dna probes for the identification of the taenia solium and taenia saginata species. |
| US6300077B1 (en) * | 1996-08-14 | 2001-10-09 | Exact Sciences Corporation | Methods for the detection of nucleic acids |
| US6268136B1 (en) * | 1997-06-16 | 2001-07-31 | Exact Science Corporation | Methods for stool sample preparation |
| AU4934099A (en) * | 1998-08-04 | 2000-02-28 | Center For Advanced Science And Technology Incubation, Ltd. | Method for enzymatic amplification of nucleic acid |
| DE19900638C2 (en) * | 1999-01-11 | 2002-12-19 | Max Planck Gesellschaft | Method of isolating DNA from biological materials |
| JP4599684B2 (en) * | 2000-07-26 | 2010-12-15 | 株式会社島津製作所 | Nucleic acid purification method from feces |
| US6852495B2 (en) * | 2002-06-06 | 2005-02-08 | Shimadzu Corporation | Process of extracting nucleic acid and process of simultaneously carrying out extraction and purification of nucleic acid |
| US7531163B2 (en) * | 2003-05-16 | 2009-05-12 | Institute For Environmental Health, Inc. | Enrichment methods for the detection of pathogens and other microbes |
| WO2005005659A1 (en) * | 2003-07-14 | 2005-01-20 | Statens Serum Institut | Diagnostics of diarrheagenic escherichia coli (dec) and shigella spp. |
| ITMI20040167A1 (en) * | 2004-02-04 | 2004-05-04 | Univ Padova | METHOD FOR SIMULTANEOUS EXTRACTION OF NUCLEIC ACIDS FROM A BIOLOGICAL SAMPLE |
| PE20061394A1 (en) * | 2005-03-15 | 2006-12-15 | Bristol Myers Squibb Co | METABOLITES OF N- (2-CHLORO-6-METHYLPHENYL) -2 - [[6- [4- (2-HYDROXYETHYL) -1-PIPERAZINYL] -2-METHYL-4-PYRIMIDINYL] AMINO] -5-THIAZOLCARBOXAMIDES |
| US7713715B2 (en) * | 2005-09-06 | 2010-05-11 | University Of Tennessee Research Foundation | Method for diagnosing infections |
| JP5566111B2 (en) * | 2007-11-20 | 2014-08-06 | オリンパス株式会社 | RNA recovery method |
-
2009
- 2009-07-23 EP EP09800428A patent/EP2314677A4/en not_active Withdrawn
- 2009-07-23 CN CN2009801285392A patent/CN102105583A/en active Pending
- 2009-07-23 WO PCT/JP2009/063163 patent/WO2010010914A1/en active Application Filing
- 2009-07-23 JP JP2010521731A patent/JP5710969B2/en not_active Expired - Fee Related
-
2011
- 2011-01-24 US US13/012,442 patent/US20110183332A1/en not_active Abandoned
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110291197A (en) * | 2016-10-27 | 2019-09-27 | 基因复制有限公司 | Detection method |
| CN110291197B (en) * | 2016-10-27 | 2024-01-12 | 基因复制股份有限公司 | Detection method |
| CN112501157A (en) * | 2020-11-30 | 2021-03-16 | 广东军融科创科技有限公司 | Kit for extracting DNA from human excrement sample, extraction method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| JPWO2010010914A1 (en) | 2012-01-05 |
| WO2010010914A1 (en) | 2010-01-28 |
| EP2314677A4 (en) | 2011-12-14 |
| EP2314677A1 (en) | 2011-04-27 |
| US20110183332A1 (en) | 2011-07-28 |
| JP5710969B2 (en) | 2015-04-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8754204B2 (en) | Method for preparing stool sample, solution for preparing stool sample, and kit for collecting stool | |
| AU2016383317B2 (en) | Molecular detection/diagnosis reagent for tumor | |
| US8597954B2 (en) | Stool sample processing method and stool sample processing container | |
| JP4264133B2 (en) | Methods for purifying, stabilizing or isolating nucleic acids from biological material | |
| US6406857B1 (en) | Methods for stool sample preparation | |
| CN102131928A (en) | Preparation method of stool sample, solution for preparation of stool sample, and kit for stool collection | |
| CN102105583A (en) | Method for collection of nucleic acid from fecal sample, method for analysis of nucleic acid, and apparatus for treatment of fecal sample | |
| WO2010064634A1 (en) | Method for preparing fecal sample, solution for preparing fecal sample, and feces collection kit | |
| JP4206425B2 (en) | Colorectal cancer marker detection method | |
| EP3800273A1 (en) | Use of detection reagent for detecting methylation of genes associated with colorectal cancer, and kit | |
| US20110236895A1 (en) | Method for preparing sample, solution for preparing sample and stool collection kit method for analyzing a nucleic acid | |
| WO2017101756A1 (en) | Kit for detecting colorectal cancer | |
| CN113981083B (en) | Nucleic acid combination and kit for colorectal cancer gene methylation detection | |
| JP2008256389A (en) | Fecal matter sample subjected to drying treatment, preparation method of fecal matter sample, and method for extracting and detecting nucleic acid or protein using fecal matter sample | |
| US20120064525A1 (en) | Method for collection of nucleic acid derived from mammalian cell, method for analysis of nucleic acid, and kit for collection of feces | |
| US20120100542A1 (en) | Method for detection of target nucleic acid, and method for testing for colon cancer | |
| CN116042605A (en) | Method and kit for extracting human intestinal wall epithelial cell DNA from feces | |
| JP2025151531A (en) | How to detect apple snails | |
| WO2008075790A2 (en) | Method for detecting cancer with nucleic acids in blood |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110622 |